Biological markers and diagnostic tests for angiotensin converting enzyme inhibitor-and vasopeptidase inhibitor-associated angioedema

ABSTRACT

Deficiencies in certain physiological pathways are linked with ACE or vasopeptidase inhibitor associated angioedema. Additionally, detection and/or measurement of dipeptidyl peptidase IV (DPP IV) enzyme activity and aminopeptidase P (APP) enzyme activity is a predictor of this risk. The present invention provides biological markers, diagnostic tests, and pharmaceutical indications that are useful in the diagnosis and treatment of angioedema and in the marketing and safety of certain medications. This ability can be important for the treatment of a subject that is in need of or are taking an angiotensin-converting enzyme (ACE) inhibitor and/or a vasopeptidase inhibitor (combined ACE and neutral endopeptidase (NEP) inhibitor), which are commonly used in the treatment of hypertension (high blood pressure), diabetes, and cardiac and renal diseases.

GRANT STATEMENT

[0001] This invention was made with federal grant money under NIH grantsHL56963, GM 07569 and 5M01 RR-00095. Thus, the United States Governmenthas certain rights in the present invention.

CROSS REFERENCE TO RELATED APPLICATIONS

[0002] The present patent application is based on and claims priority toU.S. Provisional Application Serial No. 60/244,524, entitled “BiologicalMarkers and Diagnostic Tests for Angiotensin Converting Enzyme Inhibitorand Vasopeptidase Inhibitor Associated Angioedema”, which was filed Oct.31, 2000 and is incorporated herein by reference.

TECHNICAL FIELD

[0003] The present invention relates generally to screening tests todetermine which patients are at risk for developing angioedemaassociated with inhibitors of angiotensin converting enzyme (ACE) and/orcombined ACE and neutral endopeptidase (NEP) inhibitors (a combinedACE/NEP inhibitor is referred to herein as a “vasopeptidase inhibitor”).More particularly, the present invention relates to an associationbetween dipeptidyl peptidase IV (DPP IV) and aminopeptidase P (APP)enzymatic activity and ACE and vasopeptidase inhibitor-relatedangioedema. The present invention also provides screening tests and kitsto identify a subject who is at risk for ACE and vasopeptidaseinhibitor-associated angioedema. Abbreviations ACE angiotensinconverting enzyme ACEI angiotensin converting enzyme inhibitor AGTangiotensinogen ANP atrial natriutetic peptide APP aminopeptidase P DPPIV dipeptidyl peptidase IV HTN hypertensive NCBI National Center forBiotechnology Information NEP neutral endopeptidase NLM National Libraryof Medicine NTN normotensive OMIM Online Mendelian Inheritance in ManRAS renin-angiotensin system

BACKGROUND ART

[0004] Administration of angiotensin-converting enzyme (ACE) inhibitorsis common medical practice for the treatment of a variety of diseaseconditions, including: cardiac and renal diseases, diabetes, andhypertension (high blood pressure). Several combined ACE and neutralendopeptidase (NEP) inhibitors are presently under investigation or areawaiting regulatory approval for the treatment of the aforementioneddisease conditions. However, the administration of an ACE and/or avasopeptidase inhibitor (referred to herein as an ACE/vasopeptidaseinhibitor) is contraindicated for subjects with a history of angioedemadue to the potential severity of this side effect, which can be sosevere as to result in death. Approximately 0.1% to 1.0% of thepopulation receiving an ACE inhibitor is predicted to be susceptible todeveloping at least one episode of angioedema during treatment. Thispercentage might be even higher, especially for subjects taking avasopeptidase inhibitor. Also, these inhibitors are often administeredover long periods of time because the illnesses that they treat areoften chronic conditions. This could increase the chances of a subjectdeveloping angioedema over a course of treatment.

[0005] Angioedema is an uncommon, but serious, side effect of ACE andvasopeptidase inhibitors. Currently, it is not possible to accuratelypredict which subjects are at risk to develop angioedema when taking anACE or vasopeptidase inhibitor; however it is known that approximately0.1% to 1.0% or more of the subjects receiving an ACE or vasopeptidaseinhibitor will develop angioedema as a side effect. The variation insusceptibility to vasopeptidase-associated angioedema depends, in part,on the subgroup of the population that is analyzed. For example, AfricanAmericans are particularly susceptible to ACE inhibitor associatedangioedema.

[0006] In patients who develop angioedema while taking one of thesemedications, it is difficult to determine if the angioedemic conditionarose in response to the medication or due to some other occurrence. Forexample, certain allergic reactions can result in angioedema. Thecurrent standard in practice is to employ a treatment other than anACE/vasopeptidase inhibitor, if a patient has a known history ofangioedema, or to halt treatment with ACE/vasopeptidase inhibitors if apatient presents with symptoms of angioedema or it is learnedafter-the-fact that the patient has a history of angioedema. Mostpractitioners, however, consider these alternative therapies to be lesseffective in treating the original condition than ACE/vasopeptidaseinhibitor therapy.

[0007] What is needed, therefore, are tests, assays, and biologicalmarkers for identifying patients that are at increased risk fordeveloping angioedema related to treatment with ACE/vasopeptidaseinhibitors, as compared to the general population or a matchedpopulation. Such assays would allow the continued use ofACE/vasopeptidase inhibitors in subjects that have a reducedsusceptibility to angioedema and the rational regulation of their use insusceptible subjects. The present invention solves these and otherproblems, in part by providing biological markers and diagnostic testsand kits that are preferably employed early on in treatment, therebyaverting complications.

SUMMARY OF THE INVENTION

[0008] A method of identifying a subject that is susceptible todeveloping an angioedemic condition during a course of treatmentcomprising administering one of an ACE inhibitor and a vasopeptidaseinhibitor is disclosed. In a preferred embodiment, the method comprises(a) providing a biological sample obtained from a subject; (b)determining a dipeptidyl peptidase IV activity in the biological sample;and (c) comparing a dipeptidyl peptidase IV activity in the biologicalsample to a standard dipeptidyl peptidase IV activity, wherein a 10% ormore reduction in the sample activity compared to the standard indicatesthat the subject is susceptible to developing an angioedema during acourse of treatment comprising administering one of an ACE inhibitor anda vasopeptidase inhibitor. Preferably, the vasopeptidase inhibitor is anangiotensin-converting enzyme inhibitor or a neutral endopeptidaseinhibitor. It is also preferable that a 20% or more reduction in thesample activity compared to the standard indicates that the subject issusceptible and that the subject is a human.

[0009] A method of identifying a subject that is susceptible todeveloping an angioedemic condition during a course of treatmentcomprising administering one of an ACE inhibitor and a vasopeptidaseinhibitor is disclosed. In a preferred embodiment, the method comprises:(a) providing a biological sample obtained from a subject; (b)determining an aminopeptidase P activity in the biological sample; and(c) comparing an aminopeptidase P activity activity in the biologicalsample to a standard aminopeptidase P activity, wherein a 10% or morereduction in the sample activity compared to the standard indicates thatthe subject is susceptible to developing an angioedema during a courseof treatment comprising administering one of an ACE inhibitor and avasopeptidase inhibitor. Preferably, the vasopeptidase inhibitor is anangiotensin-converting enzyme inhibitor or a neutral endopeptidaseinhibitor. It is also preferable that a 20% or more reduction in thesample activity compared to the standard indicates that the subject issusceptible and that the subject is a human.

[0010] A method of determining contraindication for administration ofone of an ACE inhibitor and a vasopeptidase inhibitor to an individualis disclosed. In a preferred embodiment, the method comprises: (a)providing a biological sample obtained from a subject; (b) determining adipeptidyl peptidase IV activity in the biological sample; and (c)comparing a dipeptidyl peptidase IV activity in the biological sample toa standard dipeptidyl peptidase IV activity, wherein administration ofthe vasopeptidase inhibitor is contraindicated when the dipeptidylpeptidase IV activity in the biological sample is outside the standarddipeptidyl peptidase IV activity range.

[0011] A method of determining contraindication for administration ofone of an ACE inhibitor and a vasopeptidase inhibitor to an individualis disclosed. In a preferred embodiment, the method comprises: (a)providing a biological sample obtained from a subject; (b) determiningan aminopeptidase P activity in the biological sample; and (c) comparingan aminopeptidase P activity in the biological sample to a standardaminopeptidase P activity, wherein administration of the vasopeptidaseinhibitor is contraindicated when the aminopeptidase P activity in thebiological sample is outside the standard aminopeptidase P activityrange.

[0012] A method of screening an individual for compatibility with anadministration of one of an ACE inhibitor and a vasopeptidase inhibitoris disclosed. In a preferred embodiment, the method comprises: (a)providing a biological sample obtained from a subject; (b) determining adipeptidyl peptidase IV activity in the biological sample; and (c)comparing a dipeptidyl peptidase IV activity in the biological sample toa standard dipeptidyl peptidase IV activity range, whereinadministration of the vasopeptidase inhibitor is contraindicated whenthe sample activity is outside the standard dipeptidyl peptidase IVactivity range, and wherein administration of the vasopeptidaseinhibitor is indicated when the sample activity is either within orabove the standard dipeptidyl peptidase IV activity range. Preferably,the vasopeptidase inhibitor is an angiotensin-converting enzymeinhibitor or a neutral endopeptidase inhibitor.

[0013] A method of screening an individual for compatibility with anadministration of one of an ACE inhibitor and a vasopeptidase inhibitoris disclosed. In a preferred embodiment, the method comprises (a)providing a biological sample obtained from a subject; (b) determiningan aminopeptidase P activity in the biological sample; and (c) comparingan aminopeptidase P activity in the biological sample to a standardaminopeptidase P activity range, wherein administration of avasopeptidase inhibitor is contraindicated when the sample activity isbelow the standard aminopeptidase P activity range, and whereinadministration of the vasopeptidase inhibitor is indicated when thesample activity is either equal to or above the standard aminopeptidaseP activity range. Preferably, the vasopeptidase inhibitor is anangiotensin-converting enzyme inhibitor or a neutral endopeptidaseinhibitor.

[0014] A kit for identifying a subject at risk for angioedema during acourse of treatment comprising administering one of an ACE inhibitor anda vasopeptidase inhibitor is disclosed. In a preferred embodiment, thekit comprises: (a) a substrate of a dipeptidyl peptidase IV enzyme; (b)a buffer; (c) a reaction stop solution; and (d) a set of instructionscomprising information on a standard dipeptidyl peptidase IV activityrange. Preferably, the article of manufacture further comprises acalibration solution for calibration of the reaction and the substrateis Gly-Pro-p-nitroanilide.

[0015] A kit for identifying a subject at risk for angioedema during acourse of treatment comprising administering one of an ACE inhibitor anda vasopeptidase inhibitor is disclosed. In a preferred embodiment, thekit comprises: (a) an aminopeptidase P enzyme substrate; (b) a dilutionbuffer; (c) a reaction stop solution; (d) a revelation buffer; and (e) aset of instructions comprising information on a standard aminopeptidaseP activity range. Preferably, the article of manufacture furthercomprises a calibration solution for calibration of the reaction and thesubstrate is the peptide Arg-Pro-Pro.

[0016] A kit for identifying a subject at risk for angioedema during acourse of treatment comprising administering one of an ACE inhibitor anda vasopeptidase inhibitor is disclosed. In a preferred embodiment, thekit comprises (a) a vasopeptidase inhibitor; and (b) a packagingmaterial comprising information that the vasopeptidase inhibitor iscontraindicated for individuals with a serum dipeptidyl peptidase IVenzyme activity outside a standard dipeptidyl peptidase IV activityrange.

[0017] A kit for identifying a subject at risk for angioedema during acourse of treatment comprising administering one of an ACE inhibitor anda vasopeptidase inhibitor is disclosed. In a preferred embodiment, thekit comprises (a) a vasopeptidase inhibitor; and (b) a packagingmaterial comprising information that the vasopeptidase inhibitor iscontraindicated for individuals with a serum aminopeptidase P enzymeactivity outside a standard aminopeptidase P activity range.

[0018] Another kit is disclosed and in a preferred embodiment comprisesa vasopeptidase inhibitor and a packaging material, wherein thepackaging material includes information that the vasopeptidase inhibitoris contraindicated for individuals with a dipeptidyl peptidase IV enzymeactivity below a normal range or is indicated for individuals with adipeptidyl peptidase IV enzyme activity within a normal range.

[0019] Another kit is disclosed and in a preferred embodiment comprisesa vasopeptidase inhibitor and a packaging material, wherein thepackaging material includes information that the vasopeptidase inhibitoris contraindicated for individuals with an aminopeptidase P enzymeactivity below a normal range or is indicated for individuals with anaminopeptidase P enzyme activity within a normal range.

[0020] A method of marketing a vasopeptidase inhibitor is disclosed andin a preferred embodiment, the method comprises providing informationabout a diagnostic test adapted to identify a subject that issusceptible to angioedema as a result of taking the vasopeptidaseinhibitor during a course of treatment comprising administering one ofan ACE inhibitor and a vasopeptidase inhibitor. Preferably, thevasopeptidase inhibitor is an angiotensin-converting enzyme inhibitor,the diagnostic test comprises detecting an activity of a dipeptidylpeptidase IV enzyme or an aminopeptidase P enzyme in a biological samplefrom the subject, and the subject is a human. It is also preferable thatthe vasopeptidase inhibitor is a neutral endopeptidase inhibitor thatthe diagnostic test includes detecting an activity of a dipeptidylpeptidase IV enzyme or an aminopeptidase P enzyme in a biological samplefrom the subject, and the subject is a human.

[0021] Accordingly, it is an object of the present invention to providea novel method and article for identifying a subject that is susceptibleto developing an angioedemic condition during a course of treatmentcomprising administering one of an ACE inhibitor and a vasopeptidaseinhibitor. This and other objects are achieved in whole or in part bythe present invention.

[0022] An object of the invention having been stated hereinabove, otherobjects will be evident as the description proceeds, when taken inconnection with the accompanying Drawings and Laboratory Examples asbest described hereinbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1 is a diagram depicting an overview of selected portions ofthe renin-angiotensin system (RAS) and a Substance P metabolic pathway.

[0024]FIG. 2 is a diagram depicting an overview ofangiotensin-converting enzyme (ACE) inhibitor and neutral endopeptidase(NEP) inhibitor action on the systems/pathways described in FIG. 1.

[0025]FIG. 3 is a diagram depicting the catalysis of angiotensin I toangiotensin II by ACE and includes the amino acid residue sequence (SEQID NOS:1 and 2) of each species and the major position for enzymaticcleavage of the angiotensin I amino acid residue chain.

[0026]FIG. 4A is a diagram depicting the catalysis of bradykinin (SEQ IDNO:3) into inactive metabolites by ACE and NEP (arrows depict the sitesof enzymatic cleavage; cleavage sites of the dipeptidyl peptidase IV(DPP IV) and aminopeptidase P (APP) pathways for the degradation ofbradykinin into inactive metabolites are indicated by dashed arrows).

[0027]FIG. 4B is a diagram depicting the catalysis of substance P (SEQID NO:4) by ACE and NEP. The arrows depict the sites of enzymaticcleavage (a cleavage site of the DPP IV pathway for the degradation ofsubstance P into inactive metabolites is indicated by a dashed arrow).

[0028]FIG. 5 is a plot depicting DDP IV activity (innanomoles/milliliter/minute or nM/ml/min) in a control population(Control), a population with ACE inhibitor (ACEI) associated angioedema(ACEI-associated), and a population treated with an ACE inhibitor butwithout angioedema (non-ACEI).

BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING

[0029] SEQ ID NO: 1 is an amino acid sequence of a peptide fragment ofangiotensin 1.

[0030] SEQ ID NO: 2 is an amino acid sequence of a peptide fragment ofangiotensin II.

[0031] SEQ ID NO: 3 is an amino acid sequence of a peptide fragment ofbradykin.

[0032] SEQ ID NO: 4 is an amino acid sequence of a peptide fragment ofsubstance P.

[0033] SEQ ID NO: 5 is a nucleotide sequence encoding human dipeptidylpeptidase IV.

[0034] SEQ ID NO: 6 is an amino acid sequence of human dipeptidylpeptidase IV.

[0035] SEQ ID NO: 7 is a nucleotide sequence encoding a soluble form ofhuman aminopeptidase P.

[0036] SEQ ID NO: 8 is an amino acid sequence of a soluble form of humanaminopeptidase P.

[0037] SEQ ID NO: 9 is a nucleotide sequence encoding a membrane-boundform of human amino peptidase P.

[0038] SEQ ID NO: 10 is an amino acid sequence of a membrane-bound formof human amino peptidase P.

DETAILED DESCRIPTION OF THE INVENTION

[0039] The present invention provides biological markers, diagnostictests, clinical assays, and articles of manufacture (such as kits usefulin the tests and assays) for identifying an increased risk fordeveloping ACE/vasopeptidase inhibitor-associated angioedema in asubject. The present invention also provides information for anappropriate course of treatment for individuals taking ACE/vasopeptidaseinhibitor medications. The articles and methods of the present inventioncan also be employed to identify a subject that has a reduced risk fordeveloping ACE/vasopeptidase inhibitor associated angioedema.

[0040] For example, by employing the articles of manufacture and methodsof the present invention, a physician can determine whether or nottreatment with an ACE/vasopeptidase inhibitor is advisable based upon arisk that the subject might develop angioedema. Likewise, a physiciancaring for a subject that has been started on an ACE/vasopeptidaseinhibitor can learn that the subject has a history of one or more eventsof angioedema unrelated to ACE or vasopeptidase inhibitor treatment. Thephysician can employ the methods of the present invention to determineif the subject is susceptible to ACE/vasopeptidase associatedangioedema. If not, or if the risk is low, then the physician cancontinue treatment with the ACE/vasopeptidase inhibitor. If the subjectis determined to be susceptible to developing ACE/vasopeptidaseinhibitor angioedema, the physician can discontinue treatment with theACE/vasopeptidase inhibitor, or can optionally select an alternativemode of treatment.

[0041] In another situation, a subject might present with angioedemawhile being treated with an ACE/vasopeptidase inhibitor. In this case,the physician typically would discontinue treatment with theACE/vasopeptidase inhibitor until the angioedemic condition is resolved.The methods and articles of the present invention can be employed todetermine whether the angioedema resulted from the administration of theACE/vasopeptidase inhibitor or if it is likely to be due to anothercause, whether defined or undefined. If the determination by the presentinvention is that the cause is not due to administration of theACE/vasopeptidase inhibitor, then the physician can restart treatmentwith an ACE/vasopeptidase inhibitor. If the determination by the presentinvention is that the cause is due to administration of theACE/vasopeptidase inhibitor (or likely due), then the physician canselect an alternative mode of treatment (ACE/vasopeptidase inhibitorsare contraindicated in this latter situation).

[0042] In another example, during the research, development, and/ormanufacture of an ACE/vasopeptidase inhibitor compounds, apharmaceutical company or other entity can employ the methods andarticles of the present invention to evaluate the safety of thecompounds. Alternatively, the entity might desire to screen testpopulations in order to identify subjects that are at increased risk ofdeveloping serious side effects, such as angioedema, associated with theadministration of the compound(s) being tested. This can make thetesting period more safe for the subjects being evaluated. Moreover, thepresent invention can reduce the possibility of negative consequencesfrom the sale of ACE/vasopeptidase inhibitors because, after aassessment performed with the methods and articles of the presentinvention, the ACE/vasopeptidase inhibitors can be contraindicated forthe populations that are most at risk.

[0043] In addition to the market for treatment of humans, ACE and/orvasopeptidase inhibitors are used to treat similar illness in pets,livestock and show animals and the methods and compositions of thepresent invention are generally applicable to these other mammals. Theoccurrence of angioedema as a side effect, even in a relatively smallfraction of the population being treated with ACE/vasopeptidaseinhibitors, has serious consequences in the marketability of these drugsand the availability of these drugs to the approximately 99% of thetreated population that does not develop angioedema.

[0044] Animals so treated can be warm-blooded vertebrates, for instance,mammals and birds. More particularly, the animal can be selected fromthe group consisting of rodent, swine, bird, ruminant, and primate. Evenmore particularly, the animal can be selected from the group consistingof a mouse, a rat, a pig, a guinea pig, poultry, an emu, an ostrich, agoat, a cow, a sheep, and a rabbit. Most particularly, the animal can bea primate, such as an ape, a monkey, a lemur, a tarsier, a marmoset, ora human.

[0045] Thus, provided is the treatment of mammals such as humans, aswell as those mammals of importance due to being endangered (such asSiberian tigers), of economical importance (animals raised on farms forconsumption by humans) and/or social importance (animals kept as pets orin zoos) to humans, for instance, carnivores other than humans (such ascats and dogs), swine (pigs, hogs, and wild boars), ruminants (such ascattle, oxen, sheep, giraffes, deer, goats, bison, and camels), andhorses. Also provided is the treatment of birds, including the treatmentof those kinds of birds that are endangered, kept in zoos, as well asfowl, and more particularly domesticated fowl, e.g., poultry, such asturkeys, chickens, ducks, geese, guinea fowl, and the like, as they arealso of economical importance to humans. Thus, provided is the treatmentof livestock, including, but not limited to, domesticated swine (pigsand hogs), ruminants, horses, poultry, and the like.

[0046] I. Definitions

[0047] Following long-standing patent law convention, the terms “a” and“an” mean “one or more” when used in this application, including theclaims.

[0048] The term “about”, as used herein when referring to a measurablevalue such as an amount of activity, weight, time, dose, etc. is meantto encompass variations of ±2%, even more preferably ±1%, and still morepreferably ±0.1% from the specified amount, as such variations areappropriate to perform the disclosed method.

[0049] As used herein, the terms “biological marker” and “biomarker” areused interchangeably and carry the meaning as understood by one ofordinary skill in the art. The term specifically encompasses a testableor measurable indicator that can be linked or associated with aphenotype or trait. The indicator can be enzymatic, genetic,biochemical, physiological, or other form as known in the art.

[0050] As used herein, the term “ACE/vasopeptidase inhibitor” means aninhibitor of ACE and/or an inhibitor of vasopeptidase. Thus, anACE/vasopeptidase inhibitor can comprise an ACE inhibitor and/or acombined ACE and NEP inhibitor.

[0051] As used herein, the term “ACE inhibitor” means an inhibitor ofangiotensin converting enzyme (ACE).

[0052] As used herein, the term “health care provider” is known in theart and specifically includes a physician, a person with authority toprescribe a medication (whether directly or indirectly), and aveterinarian. In certain embodiments, a health care provider includes anindividual that provides a medication without prescription, such as inproviding an over-the-counter medication.

[0053] As used herein, the terms “identifying subjects” and “diagnosing”are used interchangeably with regard to the detection of a“predisposition”, “increased propensity”, “risk”, “increased risk”, andthe like. The terms specifically encompass identifying the propensityfor a subject to develop ACE/vasopeptidase inhibitor associatedangioedema.

[0054] As used herein, the terms “standard”, “normal range”, “controlrange”, and “clinical range” have normal meanings as known in the art.As used herein, these terms do not apply to DPP IV or APP enzymeactivity in populations that have ACE/vasopeptidase inhibitor associatedangioedema at the time of detection or measurement. The terms “subjectrange” or “experimental range” and the like are descriptive of enzymeactivity ranges in subjects or patients with ACE/vasopeptidase inhibitorassociated angioedema (acute or in the patient history). One of ordinaryskill in the art can determine the clinical ranges for a givenpopulation and numerous clinical ranges and standards are known in theart for a variety of enzyme activities.

[0055] As used herein, the terms “vasopeptidase enzyme” and“vasopeptidase” are used interchangeably and include, but are notlimited to, angiotensin-converting enzyme (ACE) and neutralendopeptidase (NEP). Other vasopeptidases will be known to those withskill in the art.

[0056] As used herein, the term “vasopeptidase inhibitor” includes, butis not limited to, compounds that inhibit both ACE and neutralendopeptidase (NEP).

[0057] As used herein, the term “ACE/vasopeptidase inhibitor” means anACE inhibitor and/or a vasopeptidase inhibitor.

[0058] As used herein, the term “contraindicated” means a symptom orcondition that makes a treatment, procedure, or administration of amedication inadvisable.

[0059] As used herein, the terms “detecting” and “detect” are usedinterchangeably and mean qualitative and/or quantitative determinations,including measuring an amount of enzyme activity in terms of units ofactivity or units activity per unit time, and the like.

[0060] As used herein, the terms “standard dipeptidyl peptidase IVactivity” and “standard aminopeptidase P activity” mean an activity thatrepresents an average measurement of the APP and DPP IV activities of anumber of individuals. The activities can be measured by employingactivity assays such as those disclosed herein. A standard activity canbe employed as a benchmark against which an activity observed in asample is gauged.

[0061] As used herein, the term “angioedemic condition” means acondition in a subject comprising at least the onset of symptomsconsistent with a clinical diagnosis of angioedema. An angioedemiccondition can comprise symptoms and effects peripherally associated withangioedema or symptoms and effects arising as a result of the onset orpresence of angioedema.

[0062] The term “subject” as used herein refers to any invertebrate orvertebrate species. The methods of the present invention areparticularly useful in the treatment of warm-blooded vertebrates. Thus,in a preferred embodiment, the invention concerns mammals and birds.

[0063] II. General Considerations

[0064] Angiotensin-converting enzyme (ACE) inhibitors and vasopeptidaseinhibitors are indicated for the treatment of hypertension, congestiveheart failure, diabetic neuropathy, coronary artery disease, and certainother conditions. In addition, considerable research efforts are ongoingto further improve treatment of these conditions with ACE andvasopeptidase inhibitors and to identify new inhibitors. These aremedically important drugs with large markets for the treatment of humansand other mammals.

[0065] The present invention provides biological markers, diagnostictests, assays, kits, and pharmaceutical indications which are useful foridentifying individuals susceptible to developing angioedema associatedwith treatment by an angiotensin converting enzyme (ACE) inhibitor or avasopeptidase inhibitor. The markers, tests, assays, kits andindications described herein, are generally applicable to humans andother mammals.

[0066] It will be understood that the methods and articles of thepresent invention can be employed to identify subjects or individualsthat are compatible with administration of ACE/vasopeptidase inhibitors.For these subjects, ACE/vasopeptidase inhibitor treatment might beindicated depending on their need for such treatment as determined byone of ordinary skill in the art.

[0067] II.A. Angiotensin Converting Enzyme

[0068] Angiotensin-converting enzyme (ACE) catalyzes the cleavage ofangiotensin I into angiotesin II, which has an activity of raising bloodpressure (see FIG. 1). ACE and NEP catalyze the degradation ofbradykinin and substance P into inactive metabolites. NEP also catalyzesthe degradation of atrial natriutetic peptide (ANP) into inactivemetabolites. In contrast to angiotesin II, bradykinin and ANP have anactivity of lowering blood pressure. Therefore, the use oradministration of an ACE/vasopeptidase inhibitor generally results in areduction in blood pressure because these inhibitors reduce angiotesinII production and increase bradykinin and/or ANP concentrations byinhibiting their degradation into inactive metabolites (see FIG. 2).Included in the many additional applications of ACE inhibitors are thetreatment of cardiac diseases, renal diseases, and diabetes.Vasopeptidase inhibitors are also under investigation for use in theseconditions and are awaiting regulatory approval. The clinicaleffectiveness of these inhibitors might result from influences onmultiple physiological pathways, however, and the present invention isin no way bound by theory or mechanism.

[0069] The ACE enzymatic pathway is the primary pathway for angiotesinII formation and bradykinin degradation (see FIG. 3). Alternativepathways have been identified for the degradation of both bradykinin andsubstance P, however (see FIGS. 4A and 4B). These pathways comprise thedegradation of bradykinin by the aminopeptidase P (APP) and dipeptidylpeptidase IV (DPP IV) enzymes, and the degradation of substance P by DPPIV. In general, the contribution of the alternative DPP IV and APPpathways could, but not necessarily, increase during ACE/vasopeptidaseinhibition for individuals that are at a reduced risk of angioedema(“non-ACEI”) even in comparison to normotensives (“Control”, see FIG.5). On the other hand, individuals with increased angioedema risk(“ACEI-associated”) show a reduction alternative pathway activity (forexample, DPP IV).

[0070] II.B. Angiotensin Converting Enzyme and Vasopeptidase Inhibitors

[0071] As noted, ACE acts on converting angiotensin I to angiotesin II.Angiotensin II increases blood pressure and is considered a main causeof essential hypertension. A variety of studies have been directed tosubstances inhibiting ACE actions, primarily addressing the suppressionof a rise in blood pressure.

[0072] Therapeutic vasodepressors such as CAPTOPRIL™ andD-2-methyl-3-mercaptopropanoyl-L-proline have been synthesized as ACEinhibitors. Additional ACE inhibitors available commercially includeENALAPRIL™, ENALAPRILAT™, QUINAPRIL™, RAMIPRIL™, CILAZAPRIL™, DELAPRIL™,FOSENOPRIL™, ZOFENOPRIL™, INDOLAPRIL™, LISINOPRIL™, PERINDOPRIL™,SPIRAPRIL™, PENTOPRI™, PIVOPRIL™, and known pharmaceutically acceptablesalts thereof. From foodstuff, peptides having ACE inhibiting activitieshave been separated through enzymatic hydrolysis of casein (JapaneseLaid-Open Patent Publication Nos. 62-270533, 64-5497, 64-83096) andsoybean protein (Japanese Laid-Open Patent Publication Nos. 3-1671981).

[0073] Synthetic ACE inhibitors exhibit strong activities, and canexhibit adverse effects (such as angioedema). ACE inhibitory peptidesderived from casein or soybean protein have been developed withexpectation of low toxicity and high safety, even though they exhibitlow activities. Recent studies, therefore, have been focused onseparating ACE inhibitors from foodstuff materials and manufacturingthem on a large scale by chemical synthetic methods.

[0074] An ACE inhibitor derived from food protein was first reported in1979 by Oshima et al. (Oshima et al., (1979) Biochim. Biophys. Acta 556:128). Since then over 40 ACE inhibitory peptides have been disclosed todate (see, e.g., Arivoshi, (1993) Trends Food Sci. Tech., May, 1993, p.139). A number of ACE inhibitory peptides have been derived fromfoodstuff such as sour milk (Nakamura et al., (1995) J. Dairy Sci. 78:777), tuna tissue (Kohama et al., (1988) Biochem. Biophys. Res. Comm.155(1): 332), sardine muscle (Matsuda et al., (1992) Nippon NogeigakuKaishi 66(11): 1645), oyster protein (Matsumoto et al., (1994) NipponShokuhin Kogyo Gakkaishi 41(9): 589), Ficus carica (Maruyama et al.,(1989) Agric. Biol. Chem. 53(10): 2763), and rice (Muramoto & Kawamora,(1991) Food Ind. 34(11): 18). Furthermore, numerous patent applicationshave been filed in relation with ACE inhibitory peptides, includingsynthesized inhibitors as well as those isolated from natural productsSee e.g., U.S. Pat. Nos. 5,449,661; 5,071,955; 4,692,459; 4,585,758;4,512,979; 4,191,753; 3,832,337; and European Patent No. EP174162.

[0075] II.C. Angioedema

[0076] It has been observed that treatment with ACE/vasopeptidaseinhibitors is associated with the development of angioedema in a smallpercentage of individuals. The affected population accounts forapproximately 0.1% to approximately 1.0% of patients receiving treatmentwith ACE/vasopeptidase inhibitors and appears to be more prevalent amongAfrican Americans than Caucasian Americans.

[0077] In general, angioedema is a swelling of tissue and especiallyaffects the lips and other parts of the mouth, throat, larynx, eyelids,genitals, hands, and feet. Angioedema of the mouth, tongue and larynxcan be life threatening especially when severe swelling makes breathingdifficult.

[0078] The present inventor has discovered that deficiencies in thedipeptidyl peptidase IV (DPP IV) and aminopeptidase P (APP) enzymaticpathways are related to vasopeptidase inhibitor associated angioedema.For example, the present inventor discovered that DPP IV and/or APPactivity is reduced in individuals with ACE associated angioedemacompared to activity in patients with hypertension who have been treatedwith an ACE inhibitor but have not had angioedema.

[0079] III. Biological Markers

[0080] The present invention provides biological markers (also known asbiomarkers) for identifying subjects or individuals with asusceptibility to ACE/vasopeptidase inhibitor associated angioedema. Forexample, as described herein, a low DPP IV serum enzymatic activity isassociated with an increased risk that an individual will developangioedema if an ACE/vasopeptidase inhibitor is administered. In anotherexample, as described herein, a low APP serum enzymatic activity isassociated with an increased risk that an individual will developangioedema if an ACE/vasopeptidase inhibitor is administered. Thus,biological markers specifically encompasses a testable or measurableindicator that can be linked or associated with a phenotype or trait.The indicator can be enzymatic, genetic, biochemical, physiological, orother form as known in the art. Summarily, a biological marker or abiomarker demonstrates a correlation between a first condition and asecond condition.

[0081] In one aspect of the present invention, dipeptidyl peptidase IV(DPP IV) activity is a biological marker for ACE/vasopeptidase inhibitorassociated angioedema. In another aspect of the present invention,aminopeptidase P (APP) activity is a biological marker forACE/vasopeptidase inhibitor associated angioedema. In general, theactivity of either enzyme is preferably detected in a biological sampleof the subject, and more preferably a serum sample. In certainembodiments, other useful biological samples include, but are notlimited to: tissue, biopsy, interstitial fluid, feces, urine, wholeblood, and epithelium. The biological samples can be collected andprocessed according to methods known in the art for measuring enzymaticactivity (or with adaptations as would be apparent from the disclosurehereof).

[0082] In certain embodiments, the level of enzymatic activity can bemeasured qualitatively and, in other embodiments, the level of enzymaticactivity can be measured quantitatively. In certain embodiments for theevaluation of ACE/vasopeptidase inhibitor associated angioedema, DPP IVactivity can be measured and analyzed; in other embodiments APP activitycan be measured and analyzed; and in yet other embodiments, both DPP IVand APP activities can be measured and analyzed. The same is true forqualitative detection of the biological markers. Several assays aredescribed in the Examples. In general, a qualitative assay can include areaction substrate that is placed in the biological sample and reactedwith the DPP IV and/or APP enzyme present in the sample. The reactionsubstrate can change colors, for example, if the examined activity istoo low/high by a relative amount, and a color change can indicatedetection of activity. The reaction substrate can be compared to asimilar substrate preparation reacted with a control or standard. Incertain embodiments, DPP IV and/or APP enzymatic activity in abiological sample obtained from a subject can be measured in vitro andin other embodiments, it can be measured in vivo. In general, themeasured activity is inversely proportional to the risk forACE/vasopeptidase inhibitor associated angioedema. Laboratory Example 1demonstrates the use of DPP IV as a biological marker in the context ofthe present invention.

[0083] In certain embodiments, a health care professional can test asubject for risk for developing an ACE/vasopeptidase inhibitorassociated angioedema by a method comprising: detecting or measuring aserum DPP IV and/or APP activity; administering the ACE/vasopeptidaseinhibitor for a time sufficient to inhibit ACE and/or NEP activity; andthen detecting or measuring the serum DPP IV and/or APP activity again,for example, after a period of time has lapsed.

[0084] In certain aspects of this embodiment, an increase in DPP IVand/or APP activity indicates that the subject has a low risk fordeveloping ACE/vasopeptidase inhibitor associated angioedema. In certainother embodiments, a decrease in DPP IV and/or APP activity indicatesthat the subject has a high risk for developing ACE/vasopeptidaseinhibitor associated angioedema. In yet other aspects of thisembodiment, a DPP IV and/or APP activity that does not significantlychange indicates that the subject has an intermediate to high risk fordeveloping ACE/vasopeptidase inhibitor associated angioedema.

[0085] A subject's risk of developing an angioedemic condition can beanalyzed at any time, for example, when considering administering anACE/vasopeptidase inhibitor to the subject or after the administrationhas commenced. Also, the diagnostic tests described herein (which canrely on one or more biological markers) can be employed to evaluate thecause of angioedema in a patient that is currently taking anACE/vasopeptidase inhibitor.

[0086] IV. ACE Inhibitors and Vasopeptidase Inhibitors

[0087] ACE inhibitors can differ in the chemical structure of theiractive moieties, in potency, in bioavailability, in plasma half-life, inroute of elimination, in their distribution and affinity fortissue-bound ACE, and in whether they are administered as prodrugs. Thesame can be true for vasopeptidase inhibitors. Those of ordinary skillin the art recognize that the side effects of ACE inhibitors can bedivided into those that are class specific and those that relate tospecific agents. ACE inhibitors decrease systemic vascular resistancewithout increasing heart rate and they promote natriuresis. ACEinhibitors have proved effective in the treatment of hypertension. ACEinhibitors also decrease mortality in congestive heart failure and leftventricular dysfunction after myocardial infarction, and they delay theprogression of diabetic nephropathy.

[0088] Certain examples of known and commercially available ACEinhibitors are listed in Table 1. This is not meant to be an exhaustivelist, but merely exemplary of certain ACE inhibitors that can beemployed in treating subjects in need of treatment therewith. An exampleof a vasopeptidase inhibitor in development includes omapatrilat (brandname VANLEV™ by Bristol-Meyers Squibb). TABLE 1 Marketed ACE InhibitorsCompany Compound Name (Maker of Brand (Generic Drug) Brand Name Name)Captopril CAPOTEN Enalapril VASOTEC Merck Lisinopril ZESTRIL ZenecaLisinopril PRINIVIL Merck Benazepril LOTENSIN Novartis QuinaprilACCUPRIL Parke-Davis Ramipril ALTACE Monarch Trandolapril MAVIK Knoll(Roussel Uclaf) Moexipril UNIVASE Schwartz Fosinopril MONOPRIL BMSPerindep ACESRI Solva

[0089] V. ACE/Vasopeptidase Inhibitor-Associated Angioedema

[0090] ACE inhibitors have been shown to reduce mortality in patientswith congestive heart failure, diabetic nephropathy, and coronary arterydisease. In addition to ACE inhibitor-produced effects in reducingangiotensin II production, evidence from both animal studies and humanstudies indicate that cardioprotective effects of ACE inhibitors derivein part through potentiation of the effects of bradykinin (Gainer etal., (1998) New Engl. J. Med. 339: 1285-92, incorporated herein byreference). Another group of drugs have been identified with combinedACE/NEP inhibitory effects (these drugs are included in the meaning ofthe term “vasopeptidase inhibitors”), that block degradation ofbradykinin and substance P through two pathways and also block thedegradation of atrial natriutetic peptide (ANP). These combined ACE/NEPinhibitor medications appear to be particularly effect in lowering bloodpressure in hypertensive African Americans.

[0091] While it is not the inventor's desire to be bound to theory ormechanism, it is postulated that some aspect of bradykinin and/orsubstance P plays a role in potentiating angioedema (Emanueli et al.,(1998) Hypertension 31:1299-1304; Kim et al., (2000) J. Pharm. Exp.Ther. 292: 295-298; Ersahin et al., (1997) J. Cardiovasc. Pharm. 30:96-101; Blais et al., (1999) Immunopharmacology 43: 293-302; Blais etal., (1999) Peptides 20: 421-430; Damas et al., (1996) N-S Arch.Pharmacol. 354: 662-669, all of which are incorporated herein byreference). For example, an over accumulation of bradykinin and/orsubstance P might help potentiate ACE/vasopeptidase inhibitor associatedangioedema. Thus, using this example, it is postulated by the inventorthat inhibition of bradykinin and/or substance P breakdown by ACE orcombined ACE/NEP inhibitor action has beneficial effects up to a point;however, certain individuals appear to have an inability to clear anexcessive accumulation of bradykinin and/or substance P leading to anincreased risk of developing angioedema.

[0092] The risk of ACE inhibitor-associated angioedema is increased inAfrican Americans compared to Caucasians, suggesting that geneticfactors can modulate risk of angioedema (Brown et al., (1996) Clin.Pharmacol. Ther. 60: 8-13, incorporated herein by reference). Also, theinventor has observed that there is a large number of transplantrecipients among the patients with angioedema. Again, without beingbound to any theory or mechanism, the inventor hypothesizes thatcyclosporin A, which is commonly used to treat transplant patients andalso inhibits serum DPP IV activity (Scharpe et al. (1990) Clin. Chem.36: 984), results in ACE/vasopeptidase inhibitor associated angioedemain transplant recipients. Thus, a genetic and/or an acquired defect inthe aminopeptidase P and/or dipeptidyl peptidase IV pathways, whichserve as alternative pathways for the degradation of bradykinin andsubstance P, are described herein to predispose patients to thedevelopment of ACE inhibitor or vasopeptidase inhibitor angioedema.

[0093] VI. Peptide, Polypeptide and Polynucleotide Components of thePresent Invention

[0094] A variety of biological information including nucleotide andpeptide sequence information is available from public databasesprovided, for example, by the National Center for BiotechnologyInformation (NCBI) located at the United States National Library ofMedicine (NLM). The NCBI is located on the world wide web at the URL“http://www.ncbi.nim.nih.gov/” and the NLM is located on the world wideweb at the URL “http://www.nlm.nih.gov/”. The NCBI website providesaccess to a number of scientific database resources including: GenBank,PubMed, Genomes, LocusLink, Online Mendelian Inheritance in Man (OMIM),Proteins, and Structures. A common interface to the polypeptide andpolynucleotide databases is referred to as Entrez which can be accessedfrom the NCBI website on the World Wide Web at URL“http://www.ncbi.nim.nih.gov/Entrez/” or through the LocusLink website.

[0095] The following subsections disclose a plurality of molecules thatcan form an element of the present invention. This discussion is notmeant to be an inclusive list of molecules that can form a component ofthe present invention. The following subsections are included to provideadditional detail regarding components of the present invention, as wellas to help illustrate how the various molecules relate to one another invivo.

[0096] VI.A. Angiotensin I and Angiotensin II

[0097] The following summary is available in the NCBI LocusLinkdatabase:

[0098] The human AGT gene product, pre-angiotensinogen, is expressed inthe liver and is cleaved by the enzyme renin in response to loweredblood pressure. The resulting product, angiotensin I is then cleaved byangiotensin converting enzyme (ACE) to generate the physiologicallyactive enzyme [sic, peptide] angiotensin II. Human pre-angiotensinogenis encoded by two mRNAs that differ only in the length of the3′-untranslated region due to postulated use of two polyadenylationsites. There may also be alternative initiation codons (nucleotides40-42 and 67-69). AGT is involved in maintaining blood pressure and inthe pathogenesis of essential hypertension and preeclampsia.

[0099] The Homo sapiens Official Gene Symbol and Name is: AGT:angiotensinogen. In a preferred embodiment of the present invention,angiotensin I comprises the amino acid sequence of SEQ ID NO: 1. Thehormone angiotesin II is recognized as one of the most potentvasopressor agents that produces hypertension in mammals. The action ofthe enzyme renin on the plasma protein substrate angiotensinogen resultsin the production of an inactive decapeptide, angiotensin 1, which uponconversion by the non-selective angiotensin converting enzyme (ACE)provides angiotesin II, the active hormone. See e.g., Regoli et al.,(1974) Pharm. Rev. 26: 69.

[0100] Angiotensin II causes vasoconstriction and stimulates aldosteronesecretion (from the adrenal gland) that results in a rise of both bloodvolume and pressure. Inhibitors of angiotesin II are therefore useful intreating hypertension, congestive heart failure, renal insufficiencyassociated with diabetic or hypertensive nephropathy, and glaucoma. Seee.g., Garrison et al., in The Pharmacological Basis of Therapeutics, 8thEdition, (Gilman, Goodman, Rall, Nies, and Taylor, eds), Pergamon Press,New York, 1990: p. 761-762; and Dzau, (1991) New Engl. J. Med. 324:1124-1130.

[0101] Angiotensin II also can act on other organs such as the brain(Fitzsimmons, (1980) Rev. Physiol. Biochem. Pharmacol. 87: 117).Antagonists of angiotesin II are therefore useful in enhancing cognitiveperformance in patients affected by conditions such as age associatedmental impairment or Alzheimer's disease, and in treating cognitivedisorders such as anxiety. See e.g., Dennes et al., (1992) Brit. J.Pharmacol. 105: 88; and Barnes et al., (1991) FASEB J., 5: 678.

[0102] In addition, angiotesin II acts on a variety of glandular tissuesincluding the kidney, liver, and ovaries. Antagonists of angiotesin IIare useful in treating conditions, disorders, or diseases of thesetissues associated with excessive or unregulated angiotesin II activity.Antagonists of angiotesin II are also useful in treating kidney damagedue to non-steroidal antiinflammatory agents.

[0103] Angiotensin II has a role in regulation of the rate of cellgrowth and differentiation. Inhibitors of angiotesin II are thereforeuseful in treating disorders marked by excessive cell proliferation suchas restenosis. See, e.g., Naftilan et al., (1989) J. Clin. Invest. 83:1419, Kauffman et al., (1991) Life Sci. 49: 223-228, and Jackson et al.,(1988) Nature 335: 437. Angiotensin II is formed in the human bodythrough proteolysis of angiotensin I (Ang I) primarily through theaction of angiotensin-converting enzyme (see FIG. 1). In a preferredembodiment of the present invention, angiotesin II comprises the aminoacid sequence of SEQ ID NO: 2.

[0104] VI.B. Bradykinin

[0105] Bradykinin is a nonapeptide generated as a result of the activityof kallikreins, a group of proteolytic enzymes present in most tissuesand body fluids, on kininogens. Once released, kinins produce manyphysiological responses, including pain and hyperanalgesia bystimulating C- and A-fibers in the periphery. There is also considerableevidence that kinins contribute to the inflammatory response.

[0106] Bradykinin, and its physiologically important related peptideskallidin (Lys-bradykinin) and Met-Lys-bradykinin, exhibit physiologicalactions which qualify them as mediators of inflammatory reactions,hypotensive states, and pain. Bradykinin is overproduced in pathologicalconditions such as septic shock, anaphylaxis, rhinitis, asthma,inflammatory bowel disease, and certain other conditions including acutepancreatitis, post-gastrectomy dumping syndrome, carcinoid syndrome,migraine, and angioneurotic edema. The production of bradykinin from theplasma results in pain at the site of the pathological condition, andthe overproduction intensifies the pain directly or viabradykinin-induced activation of the arachidonic acid pathway whichproduces prostaglandins and leukotrienes, the more distal and actualmediators of inflammation.

[0107] In addition to its analgesic and proinflammatory effects,bradykinin is a vasodilator. Because of its ability to lower bloodpressure, bradykinin has been implicated in the pathogenesis of severalshock syndromes, particularly septic or endotoxic shock. Bradykinin isalso a potent bronchoconstrictor in animals and asthmatic subjects andit has been implicated as a contributor to the pathogenesis of airwayinflammatory conditions such as allergic asthma and rhinitis. In apreferred embodiment of the present invention, bradykinin comprises theamino acid sequence of SEQ ID NO: 3

[0108] Summarily, bradykinin increases vascular permeability, dilatesblood vessels, increases blood flow, contracts non-vascular smoothmuscle (e.g., bronchial), stimulates pain, and lowers blood pressure(hypotensive). These are also cardinal signs of inflammation. Bradykininis formed by the cleavage of kininogen by the enzyme kallikrein, and israpidly cleared in the mammalian body by cleavage into inactivemetabolites (see FIG. 1) primarily by angiotensin-converting enzyme(ACE) and neutral endopeptidase (NEP).

[0109] VI.C. Substance P

[0110] Substance P is a naturally occurring undecapeptide belonging tothe tachykinin family of peptides, the latter being so-named because oftheir prompt stimulatory action on smooth muscle tissue. More specially,substance P is a pharmaceutically active neuropeptide that is producedin mammals (having originally been isolated from gut) and possesses acharacteristic amino acid sequence that is illustrated by Veber et al.in U.S. Pat. No. 4,680,283. The wide involvement of substance P andother tachykinins in the pathophysiology of numerous diseases has beenamply demonstrated in the art. For instance, substance P has recentlybeen shown to be involved in the transmission of pain or migraine, aswell as in central nervous system disorders such as anxiety andschizophrenia, in respiratory and inflammatory diseases such as asthmaand rheumatoid arthritis, respectively, and in gastrointestinaldisorders and diseases of GI tract, like ulcerative colitis and Crohn'sdiseases, etc. It is also reported that the tachykinin antagonists areuseful for the treatment of allergic conditions, immunoregulation,vasodilation, bronchospasm, reflex or neuronal control of the visceraand senile dementia of the Alzheimer's type, emesis, sunburn andHelicobacterpylori infection.

[0111] Substance P is similar to bradykinin in function in thatsubstance P stimulates: smooth muscle contraction, inflammation, andblood vessel dilation. Substance P also functions in neurotransmission,histamine release, and activation of the immune system. Substance P issynthesized in neurons and, similar to bradykinin, is degraded intoinactive metabolites by ACE and NEP. In a preferred embodiment of thepresent invention, substance P comprises the amino acid sequence of SEQID NO: 4.

[0112] VI.D. Dipeptidyl Peptidase IV (DPP IV)

[0113] Dipeptidyl peptidase IV (DPPIV) is a serine protease that cleavesN-terminal dipeptides from a peptide chain containing, preferably, aproline residue in the penultimate position. Although the biologicalrole of DPP-IV in mammalian systems has not been completely established,it is believed to play an important role in neuropeptide metabolism,T-cell activation, attachment of cancer cells to the endothelium, andthe entry of HIV into lymphoid cells.

[0114] Various types of dipeptidyl peptidase IV have been purified andthe enzymological properties have been revealed. For example, thedipeptidyl peptidase IV is isolated from rat liver (Hopsu-Havu &Glenner, (1966) Histochem. 7: 197-201), swine kidney (Barth et al.,(1974) Acta Biol. Med. Chem. 32:157-174), small intestine (Svensson etal., (1978) Eur. J. Biochem. 90: 489-498), liver (Fukasawa et al.,(1981) Biochim. Biophys. Acta 657: 179-189), human submaxillary gland(Ova et al., (1972) Biochim. Biophys. Acta 258: 591-599), sheep kidney(Yoshimoto & Walter, (1977) Biochim. Biophys. Acta, 485: 391-401;Yoshimoto et al., (1978) J. Biol. Chem. 253: 3708-3716) ormicroorganisms (Fukusawa & Harada, (1981) Arch. Biochem. Biophys. 210:230-237; Yoshimoto & Tsuru, (1982) Biochem. 91:1899-1906).

[0115] The DPP IV enzyme is a serine exopeptidase that cleaves X-prolinedipeptides from the N-terminus of polypeptides. It is an intrinsicmembrane glycoprotein anchored into the cell membrane by its N-terminalend. Soluble forms of DPP IV are also known including those in the serum(Struvf et al., (1999) J. Immunol. 162: 4903-4909, incorporated hereinby reference). High levels of DPP IV enzyme are found in thebrush-border membranes of the kidney proximal tubule and of the smallintestine, but several other tissues also express the enzyme. DPP IVcleaves bradykinin and substance P into inactive (or reduced activity)metabolites as shown in FIGS. 4A and 4B. Table 2 discloses additionalembodiments of DPP IV. TABLE 2 Embodiments of GenBank Sequences for DPP4(DPP4 is generally referred to herein as DPP IV) Nucleotide Type ProteinAH005372 g AAB60646 U13710 g AAB60646 U13711 g AAB60646 U13712 gAAB60646 U13713 g AAB60646 U13714 g AAB60646 U13715 g AAB60646 U13716 gAAB60646 U13717 g AAB60646 U13718 g AAB60646 U13719 g AAB60646 U13720 gAAB60646 U13721 g AAB60646 U13722 g AAB60646 U13723 g AAB60646 U13724 gAAB60646 U13725 g AAB60646 U13726 g AAB60646 U13727 g AAB60646 U13728 gAAB60646 U13729 g AAB60646 U13730 g AAB60646 U13731 g AAB60646 U13732 gAAB60646 U13733 g AAB60646 U13734 g AAB60646 U13735 g AAB60646 M74777 mAAA51943 M80536 m AAA52308 X60708 m CAA43118

[0116] VI.E. Aminopeptidase P

[0117] Aminopeptidase P is known to cleave the N-terminal amino acidfrom peptides that have a prolyl residue in the second position (Orawskiet al., (1987) Mol. Cell. Biochem. 75: 123-132; Simmons & Orawski,(1992) J. Biol. Chem. 267: 4897-4903; Yoshimoto et al., (1994) Arch.Biochem. Biophys. 311: 28-34). It has been suggested that membrane-boundaminopeptidase P has an important role in vivo in the pulmonarydegradation of bradykinin (Ryan et al., (1994) J. Pharmacol. Exper.Thera. 269: 941-947; Ryan, (1989) Am. J. Physiol. 257: L53-L60; Orawski(1987) Mol. Cell. Biochem. 75: 123-132; Orawski, (1989) Adv. Exp. Med.Biol. 2478: 355-364; Simmons & Orawski, (1992) J. Biol. Chem. 267,4897-4903; Kitamura, (1995) Br. J. Pharmacol. 114: 6-7; Baker (1991)Cir. Shock 33: 37-47; Pesquero et al., (1992) J. Hyperten. 10:1471-1478; Pasquero et al., (1992) J. Hyperten. 10: 1479-1484) bycleaving its Arg¹-Pro² bond. It has also been suggested that otherpeptidases could also play a role in bradykinin degradation (Orawski etal., (1989) Adv. Exp. Med. Biol. 2478: 355-364).

[0118] Several embodiments of the aminopeptidase P enzyme are useful inthe present invention. Examples of useful embodiments are describedherein, but are not meant to limit the present invention.

[0119] VI.E.1. Aminopeptidase P (Aminopeptidase 1, Soluble)

[0120] One embodiment is the APP referred to by Homo sapiens OfficialGene Symbol and Name: XPNPEP1: X-prolyl aminopeptidase (aminopeptidaseP) 1, soluble. Table 3 presents an additional embodiment of APP. TABLE 3Certain GenBank Sequences for Aminopeptidase P1 Nucleotide Type ProteinAF195530 m AAF97866

[0121] VI.E.2. Aminopeptidase P (Aminopeptidase 2, Membrane-Bound)

[0122] Another useful embodiment is the APP referred to, in the NCBILocusLink database, by Homo sapiens Official Gene Symbol and NameXPNPEP2: X-prolyl aminopeptidase (aminopeptidase P) 2, membrane-bound.Table 4 presents additional embodiments of APP2. TABLE 4 Certain GenBankSequences for Aminopeptidase P2 Nucleotide Type Protein AL023653 gCAA19220 U90724 m AAB96394

[0123] VlI. Dipeptidyl Peptidase IV Activity Assay

[0124] The present invention also comprises an assay for dipeptidylpeptidase IV. In a preferred embodiment, the steps for performing theassay are as follows. Initially, samples comprising 0, 25, 50 and 100units (e.g., nM/ml) of p-nitroaniline are prepared for generating astandard curve. p-nitroaniline is a known substrate for DPP IV. Thestandard curve is generated by determining the absorbance of thestandard solutions of p-nitroaniline at 405 nm and are plotted on agraph as concentration versus absorbance.

[0125] To perform a DPP IV assay on a sample obtained from a subject(e.g., a human serum sample), 20 μl of sample is incubated with 10 μl of2 mM Gly-Pro-p-nitroanilide in 170 μM 0.1 M Tris-HCl for 1 hour. Thereaction is stopped by adding 800 μl sodium acetate (1 M, pH 4.5) andthe absorbance is measured at 405 nm. The concentration ofp-nitroaniline formed per ml per min is then calculated by employing astandard curve. The activity and/or presence of DPP IV in the sample canbe determined by comparing the observed activity with a standardactivity.

[0126] VIII. Aminopeptidase P Activity Assay

[0127] The present invention also comprises an aminopeptidase P activityassay. In a preferred embodiment, the steps for performing an APP assayare as follows. First, a calibration curve is prepared by monitoringfluorescence emission at 310 and 445 nm (excitation and emissionwavelengths, respectively) from a range of concentrations of 1-arginine(0-5 mM).

[0128] Next, a sample is provided (e.g. a human serum sample). 20 μl ofthe sample is incubated at 37° C. with 180 μl HEPES buffer containing5.6 mM Arg-Pro-Pro, yielding a final concentration of Arg-Pro-Pro of0.5mM. Arg-Pro-Pro is a known substrate for APP. After an incubationperiod of two hours, the reaction is stopped by adding 800 μl of cold,anhydrous ethanol to the reaction mixture. The mixture is thencentrifuged at 2000×g at 4° C. for 15 minutes. The supernatant isdecanted and incubated at room temperature with 3 ml of a revelationbuffer. APP activity is calculated as nmoles arginine released per minper ml of serum sample.

[0129] IX. Applications of the Present Invention

[0130] The present invention can be employed in a range of applications.Preferably, the present invention is employed in a situation in which aphysician is contemplating a course of treatment comprising an ACEinhibitor, a vasopeptidase inhibitor and combinations thereof. In thissituation, the present invention can be employed to minimize the riskthat a patient might develop an angioedemic condition.

[0131] The present invention can be employed, for example, to identify asubject that is susceptible to developing an angioedemic conditionduring a course of treatment which comprises administering an ACEinhibitor, a vasopeptidase inhibitor or, as is commonly the case, acombination thereof. The present invention can also be employed todetermine if administration of an ACE inhibitor, a vasopeptidaseinhibitor, or a combination thereof, is contraindicated for a subject.In a related application, the present invention can be employed in amethod of screening a subject for compatibility with administration of avasopeptidase. Additionally, the present invention can be marketed inthe form of diagnostic kits, which a physician or a researcher canemploy to identify a subject at risk for angioedema during a course oftreatment which comprises administering an ACE inhibitor, avasopeptidase inhibitor or, as is commonly the case, a combinationthereof. These are just a few of the range of applications in which thepresent invention can be employed. These applications are described morefully herein below and in the Examples that follow.

[0132] IX.A. Method of Identifying a Subject That is Susceptible toDeveloping an Angioedemic Condition During a Course of Treatment

[0133] An aspect of the present invention is the observation that thereis a link between DPP IV and/or APP activity, ACE and/or vasopeptidaseinhibitors and the onset of an angioedemic condition. Thus, when asubject is undergoing a course of treatment comprising administering anACE inhibitor, a vasopeptidase inhibitor or a combination thereof, it ispreferable to determine the activity of DPP IV and/or APP in a samplederived from the subject. Depressed DPP IV and/or APP activity levelsindicate that the subject is at risk for developing an angioedemiccondition as a result of the course of treatment.

[0134] In a preferred embodiment of this application of the presentinvention, a biological sample is initially provided by a subject.Preferably, the sample is a serum sample. A sample can be acquired froma subject by employing standard techniques, and will be dependent, inpart, on the nature of the sample. For example, when the samplecomprises a sample of the subject's blood, standard phlebotomic methodscan be employed to acquire the sample, which can be further processed asrequired (e.g. isolating a serum component of sample).

[0135] Following sample acquisition and preparation (if required), astandard DPP IV and/or APP activity is determined. A standard DPP IVand/or APP activity can be determined by calculating DPP IV and/or APPactivity in a control group of subjects. The number of subjects canvary, but preferably, the number of subjects is sufficiently large as topermit the identification of significant activity measurement.Similarly, the genetic qualities of the subjects can vary or can be heldconstant, at the preference of the researcher. This calculated activitycan be employed as a standard (i.e. a standard DPP IV and/or APPactivity), against which a subject's determined DPP IV and/or APPactivity is gauged.

[0136] Subsequently, DPP IV and/or APP activity present in the samplecan be determined. Both a standard DPP IV and/or APP, as well as DPP IVand/or APP activity present in a sample, can be measured by employing,for example, the activity assays disclosed herein, particularly insection VII (DPP IV activity) and in section VIII (APP activity).

[0137] When a value is determined for both a standard DPP IV and/or APPactivity and DPP IV and/or APP activity present in a sample, the twovalues can be compared. If DPP IV and/or APP activity in the sample isfound to be less than the activity of the control group (i.e., astandard activity) by about 10% or more, the subject is at risk for anangioedemic condition, should ACE and/or vasopeptidase inhibitor therapybe started or continued. Thus, ACE and/or vasopeptidase inhibitortherapy is contraindicated for subjects in which the DPP IV and/or APPactivity of a sample is found to be less than the activity of thecontrol group (i.e., a standard activity) by about 10% or more. On theother hand, ACE and/or vasopeptidase inhibitor therapy can be toleratedand/or indicated for subjects in which the DPP IV and/or APP activity ofa sample is found to be within about 10% or less of the activity of thecontrol group (i.e. a standard activity).

[0138] A 20% or more reduction in the DPP IV and/or APP activity in thebiological sample, as compared to the standard DPP IV and/or APPactivity also indicates that the subject is susceptible to developing anangioedema during a course of treatment comprising administering one ofan ACE inhibitor and a vasopeptidase inhibitor. Additionally, a 30% ormore reduction in the DPP IV and/or APP activity in the biologicalsample, as compared to the standard DPP IV and/or APP activity indicatesthat the subject is susceptible to developing an angioedema during acourse of treatment comprising administering one of an ACE inhibitor anda vasopeptidase inhibitor.

[0139] IX.B. Method of Determining Contraindication for Administrationof a Vasopeptidase Inhibitor, an ACE Inhibitor and Combinations Thereof

[0140] In another aspect of the present invention, a vasopeptidateinhibitor, an ACE inhibitor and combinations thereof can becontraindicated if DPP IV and/or APP activity is found to fall outsidethe range of normal activities and/or amounts. APP and DPP IV activitiescan be determined by employing the assays disclosed in the presentinvention. In a preferred method of determining contraindication foradministration of an ACE inhibitor or a vasopeptidase inhibitor to anindividual, a biological sample obtained from a subject is initiallyprovided. Preferably, the biological sample comprises serum and isobtained from a human subject, although the method can also be performedin the context of an organism other than a human and a sample cancomprise a material other than serum.

[0141] Next, a standard DPP IV activity and/or APP activity can bedetermined and can be plotted to generate a standard curve. The standardDPP IV activity and/or APP activity can be determined by measuring a DPPIV and/or APP activity from a number of representative subjects. Astandard DPP IV activity and/or APP activity measurement can serve as abenchmark against which a DPP IV activity and/or APP activity observedin a sample is measured.

[0142] Following providing (and preparing, if desired) a biologicalsample, a DPP IV activity and/or a APP activity for the biologicalsample can be determined. The DPP IV and/or APP activities can bedetermined as disclosed herein, and are preferably performed under thesame conditions as were employed in generating the standard activity(i.e. the standard curve).

[0143] Observed DPP IV activity and/or APP activity in the biologicalsample can then be compared to the standard DPP IV activity and or APPactivity. If the comparison indicates that DPP IV and/or APP activity inthe biological sample is below the normal range, administration of anACE or a vasopeptidase inhibitor can be contraindicated.Contraindication of administration of an ACE inhibitor or avasopeptidase inhibitor can impart the beneficial effect of decreasingor eliminating the chance that a subject will develop an angioedemiccondition.

[0144] IX.C. A Kit For Identifying a Subject at Risk for AngioedemaDuring a Course of Treatment Comprising Administering an ACE Inhibitor,a Vasopeptidase Inhibitor or a Combination Thereof

[0145] In another aspect of the present invention, a kit for identifyinga subject at risk for angioedema during a course of treatment comprisingadministering an ACE inhibitor, a vasopeptidase inhibitor or acombination thereof is disclosed. Such a kit can be employed by aphysician, laboratory researcher or other person desiring to identify anindividual at risk for developing an angioedemic condition. In apreferred embodiment, the kit comprises a substrate for a DPP IV enzyme.Such a substrate preferably comprises gly-pro-p-nitroanilide; however,other substrates can be employed.

[0146] A kit of the present invention also preferable comprises abuffer, which can function to maintain pH and other conditions in anoptimal range for a DPP assay. Any buffer adapted to maintain a set ofdesired conditions (e.g., pH, tonicity, etc) can be employed in a kit. Areaction stop solution is also preferably included in the kit. Thereaction stop solution can be added to a reaction mixture in order tohalt any DPP IV-catalyzed reaction occurring in the reaction mixture ata desired time point.

[0147] Additionally, a kit preferably comprises a set of instructionscomprising information on a range of dipeptidyl peptidase IV activity ina control population. The information contained in such a set ofinstructions can advise a physician or researcher (or any person) who isemploying the kit on the question of how to compare a DPP IV activityobserved in a sample with a standard DPP IV activity. In other words, aset of instructions can advise the user of the kit how to interpret theresults of a test performed by employing the kit. A set of instructionscan also comprise step-by-step directions on how a user can employ thevarious components of the kit to generate an observed DPP IV activityfrom a sample. Thus, such a set of instructions can comprise informationon volumes of solutions to be added, incubation time periods,wavelengths to monitor (if any) and other parameters of a DPP IV assay.

[0148] In practice, if an observed DPP IV activity falls within a rangespecified in a set of instructions, administering an ACE inhibitor, avasopeptidase inhibitor or a combination thereof can be administered toa subject with the knowledge that the risk of the subject developing anangioedemic condition is minimal. Thus, such a kit can be employed toidentify a subject at risk for developing an angioedemic conditionbefore a course of treatment comprising administering a vasopeptidaseinhibitor and/or an ACE inhibitor.

[0149] In another embodiment of a kit for identifying a subject at riskfor angioedema during a course of treatment comprising administering anACE inhibitor, a vasopeptidase inhibitor or a combination thereof, thekit comprises an APP substrate. A suitable APP substrate can comprise,for example, a peptide sequence comprising Arg-Pro-Pro. A dilutionbuffer can also be included and can be used to dilute a substratesolution or other concentrated solution supplied with the kit or derivedfrom a sample acquired from a subject. A reaction stop solution can alsobe included, as well as a revelation buffer. The revelation buffer canassist in maintaining conditions under which APP activity in a samplecan be determined. For example, if a colorimetric assay is employed, arevelation buffer can be employed to develop a degree of color.Alternatively, if a spectrophotometric assay is employed, the revelationbuffer can be employed to maintain conditions under which a detectablereaction product can remain in a detectable state (i.e. undegraded).

[0150] Additionally, a set of instructions comprising information on arange of APP activity in a control population can be provided with a kitof the present invention. As described above in the context of DPP IV,if an observed APP activity falls within a range specified in a set ofinstructions, administering an ACE inhibitor, a vasopeptidase inhibitoror a combination thereof can be administered to a subject with theknowledge that the risk of the subject developing an angioedemiccondition is minimal. Thus, such a kit can be employed to identify asubject at risk for developing an angioedemic condition before a courseof treatment comprising administering a vasopeptidase inhibitor and/oran ACE inhibitor.

[0151] In yet another embodiment, a kit for identifying a subject atrisk for angioedema during a course of treatment comprisingadministering an ACE inhibitor, a vasopeptidase inhibitor or acombination comprises an ACE inhibitor and/or a vasopeptidase inhibitor;and a packaging material comprising information that the vasopeptidaseinhibitor is contraindicated for individuals with a serum DPP IV enzymeactivity and/or a serum APP enzyme activity below a normal range, whichcan be specified in the packaging material.

[0152] IX.D. Method of Marketing a Vasopeptidase and/or an ACE Inhibitor

[0153] A method of marketing a vasopeptidase and/or an ACE inhibitor isalso disclosed. In one embodiment, information about a diagnostic testadapted to identify a subject that is susceptible to angioedema as aresult of taking the vasopeptidase inhibitor during a course oftreatment comprising administering an ACE inhibitor, a vasopeptidaseinhibitor, or a combination thereof is provided. When it is known that agiven subject might be at risk for developing an angioedemic condition,this information can comprise an element of a marketing approach. Inthis way, a vasopeptidase and/or ACE inhibitor can be marketed toindividuals who can tolerate these inhibitors, while subjects that mightbe susceptible to developing an angioedemic condition as a result of acourse of treatment comprising these inhibitors can be advised of thisrisk.

[0154] This information can be presented to a consumer, whether theconsumer is a physician or a subject, at the time an inhibitor ispurchased. Alternatively, the information can be presented to a consumerat a point prior to purchase. This method of marketing can beadvantageous because it is not only a marketing tool, but can alsodecrease the risk of a subject developing an angioedemic condition.

[0155] X. Illustrative Examples of Preferred Embodiments

[0156] This section of the present disclosure provides illustrativeexamples of the application of the present invention. The IllustrativeExamples, therefore, provide additional guidance in the application ofthe present invention. These illustrative examples resemble medical casestudies, since the present invention is preferably suited to therapeuticapplication (and therefore of particular benefit to physicians), inaddition to being a valuable research tool. The Illustrative Examplesare ordered similarly; first, facts of the case are presented, andsubsequently, several outcomes are presented. These outcomes describetreatments a physician can recommend. In the Illustrative Examples, thephysician in the examples arrives at his or her recommendation as aresult of employing the present invention. In other words, the physicianorders a test, which involves various aspects of the present invention(i.e. a determination of DPP IV activity, APP activity, etc). Thephysician then evaluates the results of the test and recommends a courseof treatment. Thus, the alternative outcomes presented in theIllustrative Examples are based on the results of the test or testsordered by the physician. The Illustrative Examples, therefore, serve todemonstrate how the present invention can be employed in a clinicalsetting.

ILLUSTRATIVE EXAMPLE 1

[0157] A 55-year-old African American woman smoker with diabeticnephropathy presents to clinic with poorly controlled hypertension. Sheis taking hydrochlorothiazide alone for treatment of her hypertension.Because of the patient's diabetic nephropathy the patient's physiciandetermines that an ACE inhibitor is the drug of choice for treatment ofher high blood pressure. However, based on demographic factors, thephysician calculates that the patient's risk of ACE inhibitor-associatedangioedema is high (1:400). (One of ordinary skill in the art is able tocalculate an individual's risk based upon the scientific literature andthe race of the patients.) He therefore draws blood for measurement ofDPP IV activity and APP activity prior to starting her on an ACEinhibitor.

Outcome A of Illustrative Example 1

[0158] The patient's DPP IV and APP activities are found to be normaland she carries no genetic alleles associated with decreased activity.On this basis, the physician calculates that the patient's risk ofangioedema is lower than predicted by demographics and starts her on anACE inhibitor.

Outcome B of Example 1

[0159] The patient is found to have decreased DPP IV activity. On thisbasis her calculated risk of angioedema is unacceptably high and thephysician chooses an alternative therapy.

ILLUSTRATIVE EXAMPLE 2

[0160] A 64-year-old African American man with dilated cardiomyopathyand a history of congestive heart failure presents to the emergency roomwith swelling of his lips and oropharynx. On examination he is noted tobe stridorous and he is intubated to protect his airway. He is givenintravenous corticosteroids and histamine H₁ and H₂ antagonists. Priorto admission he was taking the diuretic furosemide, the ACE inhibitorlisinopril, and the aldosterone receptor antagonist spironolactone. Hehas taken the ACE inhibitor for at least four years and has never hadany previous episode of angioedema. Five days prior to admission he wasstarted on the antibiotic ciprofloxacin for a urinary tract infection.It was not clear to the patient's physician whether his angioedema wasrelated to his use of an ACE inhibitor. Given the proven benefit of ACEinhibitors as treatment in patients with left ventricular dysfunction,the physician desired to continue therapy, if possible. The physiciandraws blood samples for measurement of C₁ esterase inhibitor activity,APP and DPP IV activity, as well as a sample for extraction and analysisof DNA markers and sequences.

Outcome A of Illustrative Example 2

[0161] C₁ esterase inhibitor activity is found to be normal, excludingC₁ esterase inhibitor deficiency associated hereditary angioedema.However, DPP IV activity is found to be below the normal range. It isdetermined that it is not safe to restart the patient's ACE inhibitor,since the risk of angioedema is high.

Outcome B of Illustrative Example 2

[0162] The C₁ esterase inhibitor activity is found to be normal,excluding C₁ esterase inhibitor associated hereditary angioedema. TheDPP IV activity is found to be below the normal range. The physiciandetermines that treatment with the ACE inhibitor is still the bestpossible mode of treatment, once the angioedema is resolved, and thephysician wants to determine if biomarkers and biochemical indicators(e.g., DPP IV activity) reveal that the angioedema was an isolatedepisode possibly related to some other exposure. Thus, the DPP IVactivity is measured again in about 2 weeks or more after the firstmeasurement (or after the angioedema has resolved).

Outcome B1 of Illustrative Example 2

[0163] The DPP IV activity found to remain depressed even after theangioedema has resolved. The physician determines that the risk of arecurrent episode of angioedema is high and orders that theACE/vasopeptidase inhibitor treatment should not be restarted.

Outcome B2 of Illustrative Example 2

[0164] The DPP IV activity found to increase sufficiently after theangioedema has resolved or returns to normal, such that the physiciandetermines that the angioedema was related to an isolated acquiredinfluence. The physician determines that the patient's episode ofangioedema is likely related to concurrent ciprofloxacin administrationand that the risk of a recurrent episode of angioedema is low. The ACEor vasopeptidase inhibitor treatment is restarted at the original doselevel or, alternatively, at a lower dose than the original dose of ACEor vasopeptidase inhibitor.

ILLUSTRATIVE EXAMPLE 3

[0165] A physician determines that a patient is in need of treatmentwith an ACE/vasopeptidase inhibitor. A blood sample is drawn from thepatient and is processed to obtain a serum sample. The DPP IV and/or APPactivity is determined for the individual. The patient is started on theinhibitor(s). The DPP IV and/or APP enzyme activity is checkedperiodically to determine the risk for angioedema and to determine ifthe risk is changing. The period between tests can be any periodselected by the physician. In certain examples the period is about oneweek, in certain examples the period is about six months and in certainexamples the period varies from test to test.

ILLUSTRATIVE EXAMPLE 4

[0166] Example 4 is the same as Example 3, except that the patientdevelops angioedema during the course of treatment with theinhibitor(s). Treatment with the inhibitor(s) is suspended until theangioedema is resolved and until the DPP IV and/or APP enzyme activityis found to be at a safe level(s) to resume treatment with theinhibitor(s).

LABORATORY EXAMPLES

[0167] The following Laboratory Examples have been included toillustrate preferred modes of the invention. Certain aspects of thefollowing Laboratory Examples are described in terms of techniques andprocedures found or contemplated by the present inventors to work wellin the practice of the invention. These Laboratory Examples areexemplified through the use of standard laboratory practices of theinventors. In light of the present disclosure and the general level ofskill in the art, those of skill will appreciate that the followingLaboratory Examples are intended to be exemplary only and that numerouschanges, modifications and alterations can be employed without departingfrom the spirit and scope of the invention.

Laboratory Example 1

[0168] One use of DPP IV enzyme activity as a biological marker isdemonstrated in FIG. 5. In this example, DPP IV activity is in a rangeof about 28 to about 42 nM/ml/min in a control group. The control groupcomprises subjects that have received an ACE or vasopeptidase inhibitorand do not have angioedema (they are normotensive). Thus, 28 to 42nM/ml/min is considered to be the normal range or control range for thisparticular population, in this example. DPP IV activity in a group ofhypertensive subjects who have received an ACE inhibitor, but were freefrom angioedema, is in a range above the normotensive control group inthis experiment. Thus, above 28 and preferably above 40 nM/ml/min isconsidered to be the normal range or control range for this particulargroup (for example, 40 to 50 nM/ml/min; for another example 40 to morethan 40 nM/ml/min). A group receiving an ACE inhibitor and presentingwith acute angioedema has reduced DPP IV enzymatic activity. The subjectrange is between 18 and 27 nM/ml/min, in this example. Thus, this groupshows a reduction in the average and the median DPP IV activity comparedto the hypertensive group. There is a significant difference in theranges of DPP IV activity between these groups and the significance isgreater than or equal to a 95% confidence interval.

[0169] Referring now to Table 5, Column A (NTN) is normotensivecontrols. Column B (HTN) is hypertensive controls (received ACEinhibitor at some time). Column C is a subject group with acuteangioedema and receiving ACE inhibitor. Values that are outside a“range” can be outside of the 95% confidence interval, for example.TABLE 5 RESULTS OF A CLINICAL TRIAL X Labels A B C D E X Labels NTN HTNACEI AE ACEI AE non-ACEI AE X Y Y Y Y Y Number of 21 10 5 7 2 valuesMinimum 24.80 28.08 23.97 19.68 41.25 25% Percentile 34.21 30.28 31.61Median 38.06 35.41 24.61 35.57 42.06 75% Percentile 42.80 39.17 43.15Maximum 51.59 39.75 28.38 43.57 42.87 Mean 37.76 34.59 25.32 35.12 42.06Std. Deviation 6.300 4.243 1.774 8.511 1.146 Std. Error 1.375 1.3420.7935 3.217 0.8100 Lower 95% CI 34.90 31.55 23.12 27.25 31.77 Upper 95%CI 40.63 37.62 27.53 42.99 52.35

References

[0170] The references listed below as well as all references cited inthe specification are incorporated herein by reference to the extentthat they supplement, explain, provide a background for or teachmethodology, techniques and/or compositions employed herein. All citedpatents and publications referred to in this application are hereinexpressly incorporated by reference. Also expressly incorporated hereinby reference are the contents of all citations of GenBank accessionnumbers, LocusID, and other computer database listings.

[0171] Ariyoshi, (1993) Trends Food Sci. Tech., May, 1993, p. 139

[0172] Baker (1991) Cir. Shock 33: 37-47

[0173] Barth et al., (1974) Acta Biol. Med. Chem. 32:157-174

[0174] Blais et al., (1999) Immunopharmacology 43: 293-302

[0175] Blais et al., (1999) Peptides 20: 421-430

[0176] Brown et al., (1996) Clin. Pharmacol. Ther. 60: 8-13

[0177] Damas et al., (1996) N-S Arch. Pharmacol. 354: 662-669

[0178] Dennes et al., (1992) Brit. J. Pharmacol. 105: 88; and Barnes etal., (1991) FASEB J., 5: 678

[0179] Dzau, (1991) New Engl. J. Med. 324: 1124-1130

[0180] Emanueli et al., (1998) Hypertension 31:1299-1304

[0181] Ersahin et al., (1997) J. Cardiovasc. Pharm. 30: 96-101

[0182] Fitzsimmons, (1980) Rev. Physiol. Biochem. Pharmacol. 87: 117

[0183] Fukasawa et al., (1981) Biochim. Biophys. Acta 657: 179-189

[0184] Fukusawa & Harada, (1981) Arch. Biochem. Biophys. 210: 230-237

[0185] Gainer et al., (1998) New Engl. J. Med. 339: 1285-92

[0186] Garrison et al., in The Pharmacological Basis of Therapeutics,8th Edition, (Gilman, Goodman, Rail, Nies, and Taylor, eds), PergamonPress, New York, 1990: p. 761-762

[0187] Hopsu-Havu & Glenner, (1966) Histochem. 7:197-201

[0188] Jackson et al., (1988) Nature 335: 437

[0189] Kauffman et al., (1991) Life Sci. 49: 223-228

[0190] Kim et al., (2000) J. Pharm. Exp. Ther. 292: 295-298

[0191] Kitamura, (1995) Br. J. Pharmacol. 114: 6-7

[0192] Kohama et al., (1988) Biochem. Biophys. Res. Comm. 155(1): 332

[0193] Maruvama et al., (1989) Agric. Biol. Chem. 53(10): 2763

[0194] Matsuda et al., (1992) Nippon Nogeigaku Kaishi 66(11): 1645

[0195] Matsumoto et al., (1994) Nippon Shokuhin Kogyo Gakkaishi 41(9):589

[0196] Muramoto & Kawamora, (1991) Food Ind. 34(11): 18

[0197] Naftilan et al., (1989) J. Clin. Invest. 83: 1419

[0198] Nakamura et al., (1995) J. Dairy Sci. 78: 777

[0199] Orawski (1987) Mol Cel. Biochem. 75:123-132

[0200] Orawski et al., (1987) Mol. Cell. Biochem. 75: 123-132

[0201] Orawski et al., (1989) Adv. Exp. Med. BioL. 2478: 355-364

[0202] Oshima et al., (1979) Biochim. Biophys. Acta 556:128

[0203] Oya et al., (1972) Biochim. Biophys. Acta 258: 591-599

[0204] Pasguero et al., (1992) J. Hyperten. 10: 1479-1484

[0205] Pesqueroetal., (1992) J. Hyperten. 10: 1471-1478

[0206] Regoli et al., (1974) Pharm. Rev. 26: 69

[0207] Ryan et al., (1994) J. PharmacoL Exper. Thera. 269: 941-947

[0208] Ryan, (1989) Am. J. Physiol. 257: L53-L60

[0209] Scharpe et al., (1990) Clin. Chem. 36: 984

[0210] Simmons & Orawski, (1992) J. Biol. Chem. 267, 4897-4903

[0211] Struyf et al., (1999) J. Immunol. 162: 4903-4909

[0212] Svensson et al., (1978) Eur. J. Biochem. 90: 489-498

[0213] Yoshimoto & Tsuru, (1982) Biochem. 91:1899-1906

[0214] Yoshimoto & Walter, (1977) Biochim. Biophys. Acta, 485: 391-401

[0215] Yoshimoto et al., (1978) J. Biol. Chem. 253: 3708-3716

[0216] Yoshimoto et al., (1994) Arch. Biochem. Biophys. 311: 28-34

[0217] European Patent No. EP174162

[0218] Japanese Patent No. 3-1671981

[0219] Japanese Patent No. 62-270533

[0220] Japanese Patent No. 64-5497

[0221] Japanese Patent No. 64-83096

[0222] U.S. Pat. No. 3,832,337

[0223] U.S. Pat. No. 4,191,753

[0224] U.S. Pat. No. 4,512,979

[0225] U.S. Pat. No. 4,585,758

[0226] U.S. Pat. No. 4,680,283

[0227] U.S. Pat. No. 4,692,459

[0228] U.S. Pat. No. 5,071,955

[0229] U.S. Pat. No. 5,449,661

[0230] It will be understood that various details of the invention maybe changed without departing from the scope of the invention. Moreover,it is not the inventor's desire to be bound by theory or mechanism. Anytheory or mechanism presented herein is included solely to supplementthe disclosure, and should not be interpreted to impose any limitationon the claims presented hereinbelow. Therefore, the foregoingdescription is for the purpose of illustration only, and not for thepurpose of limitation—the invention being defined by the claims.

1 10 1 10 PRT Homo sapiens 1 Asp Arg Val Tyr Ile His Pro Phe His Leu 1 510 2 8 PRT Homo sapiens 2 Asp Arg Val Tyr Ile His Pro Phe 1 5 3 9 PRTHomo sapiens 3 Arg Pro Pro Gly Phe Ser Pro Phe Arg 1 5 4 11 PRT Homosapiens 4 Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Met 1 5 10 5 3407 DNAHomo sapiens 5 cgcgcgtctc cgccgcccgc gtgacttctg cctgcgctcc ttctctgaacgctcacttcc 60 gaggagacgc cgacgatgaa gacaccgtgg aagattcttc tgggactgctgggtgctgct 120 gcgcttgtca ccatcatcac cgtgcccgtg gttctgctga acaaaggcacagatgatgct 180 acagctgaca gtcgcaaaac ttacactcta actgattact taaaaaatacttatagactg 240 aagttatact ccttaagatg gatttcagat catgaatatc tctacaaacaagaaaataat 300 atcttggtat tcaatgctga atatggaaac agctcagttt tcttggagaacagtacattt 360 gatgagtttg gacattctat caatgattat tcaatatctc ctgatgggcagtttattctc 420 ttagaataca actacgtgaa gcaatggagg cattcctaca cagcttcatatgacatttat 480 gatttaaata aaaggcagct gattacagaa gagaggattc caaacaacacacagtgggtc 540 acatggtcac cagtgggtca taaattggca tatgtttgga acaatgacatttatgttaaa 600 attgaaccaa atttaccaag ttacagaatc acatggacgg ggaaagaagatataatatat 660 aatggaataa ctgactgggt ttatgaagag gaagtcttca gtgcctactctgctctgtgg 720 tggtctccaa acggcacttt tttagcatat gcccaattta acgacacagaagtcccactt 780 attgaatact ccttctactc tgatgagtca ctgcagtacc caaagactgtacgggttcca 840 tatccaaagg caggagctgt gaatccaact gtaaagttct ttgttgtaaatacagactct 900 ctcagctcag tcaccaatgc aacttccata caaatcactg ctcctgcttctatgttgata 960 ggggatcact acttgtgtga tgtgacatgg gcaacacaag aaagaatttctttgcagtgg 1020 ctcaggagga ttcagaacta ttcggtcatg gatatttgtg actatgatgaatccagtgga 1080 agatggaact gcttagtggc acggcaacac attgaaatga gtactactggctgggttgga 1140 agatttaggc cttcagaacc tcattttacc cttgatggta atagcttctacaagatcatc 1200 agcaatgaag aaggttacag acacatttgc tatttccaaa tagataaaaaagactgcaca 1260 tttattacaa aaggcacctg ggaagtcatc gggatagaag ctctaaccagtgattatcta 1320 tactacatta gtaatgaata taaaggaatg ccaggaggaa ggaatctttataaaatccaa 1380 cttattgact atacaaaagt gacatgcctc agttgtgagc tgaatccggaaaggtgtcag 1440 tactattctg tgtcattcag taaagaggcg aagtattatc agctgagatgttccggtcct 1500 ggtctgcccc tctatactct acacagcagc gtgaatgata aagggctgagagtcctggaa 1560 gacaattcag ctttggataa aatgctgcag aatgtccaga tgccctccaaaaaactggac 1620 ttcattattt tgaatgaaac aaaattttgg tatcagatga tcttgcctcctcattttgat 1680 aaatccaaga aatatcctct actattagat gtgtatgcag gcccatgtagtcaaaaagca 1740 gacactgtct tcagactgaa ctgggccact taccttgcaa gcacagaaaacattatagta 1800 gctagctttg atggcagagg aagtggttac caaggagata agatcatgcatgcaatcaac 1860 agaagactgg gaacatttga agttgaagat caaattgaag cagccagacaattttcaaaa 1920 atgggatttg tggacaacaa acgaattgca atttggggct ggtcatatggagggtacgta 1980 acctcaatgg tcctgggatc gggaagtggc gtgttcaagt gtggaatagccgtggcgcct 2040 gtatcccggt gggagtacta tgactcagtg tacacagaac gttacatgggtctcccaact 2100 ccagaagaca accttgacca ttacagaaat tcaacagtca tgagcagagctgaaaatttt 2160 aaacaagttg agtacctcct tattcatgga acagcagatg ataacgttcactttcagcag 2220 tcagctcaga tctccaaagc cctggtcgat gttggagtgg atttccaggcaatgtggtat 2280 actgatgaag accatggaat agctagcagc acagcacacc aacatatatatacccacatg 2340 agccacttca taaaacaatg tttctcttta ccttagcacc tcaaaataccatgccattta 2400 aagcttatta aaactcattt ttgttttcat tatctcaaaa ctgcactgtcaagatgatga 2460 tgatctttaa aatacacact caaatcaaga aacttaaggt tacctttgttcccaaatttc 2520 atacctatca tcttaagtag ggacttctgt cttcacaaca gattattaccttacagaagt 2580 ttgaattatc cggtcgggtt ttattgttta aaatcatttc tgcatcagctgctgaaacaa 2640 caaataggaa ttgtttttat ggaggctttg catagattcc ctgagcaggattttaatctt 2700 tttctaactg gactggttca aatgttgttc tcttctttaa agggatggcaagatgtgggc 2760 agtgatgtca ctagggcagg gacaggataa gagggattag ggagagaagatagcagggca 2820 tggctgggaa cccaagtcca agcataccaa cacgagcagg ctactgtcagctcccctcgg 2880 agaagagctg ttcaccacga gactggcaca gttttctgag aaagactattcaaacagtct 2940 caggaaatca aatatcgaaa gcactgactt ctaagtaaac cacagcagttgaaagactcc 3000 aaagaaatgt aagggaaact gccagcaacg cagcccccag gtgccagttatggctatagg 3060 tgctacaaaa acacagcaag ggtgatggga aagcattgta aatgtgcttttaaaaaaaaa 3120 tactgatgtt cctagtgaaa gaggcagctt gaaactgaga tgtgaacacatcagcttgcc 3180 ctgttaaaag atgaaaatat ttgtatcaca aatcttaact tgaaggagtccttgcatcaa 3240 tttttcttat ttcatttctt tgagtgtctt aattaaaaga atattttaacttccttggac 3300 tcattttaaa aaatggaaca taaaatacaa tgttatgtat tattattcccattctacata 3360 ctatggaatt tctcccagtc atttaataaa tgtgccttca ttttttc 34076 766 PRT Homo sapiens 6 Met Lys Thr Pro Trp Lys Ile Leu Leu Gly Leu LeuGly Ala Ala Ala 1 5 10 15 Leu Val Thr Ile Ile Thr Val Pro Val Val LeuLeu Asn Lys Gly Thr 20 25 30 Asp Asp Ala Thr Ala Asp Ser Arg Lys Thr TyrThr Leu Thr Asp Tyr 35 40 45 Leu Lys Asn Thr Tyr Arg Leu Lys Leu Tyr SerLeu Arg Trp Ile Ser 50 55 60 Asp His Glu Tyr Leu Tyr Lys Gln Glu Asn AsnIle Leu Val Phe Asn 65 70 75 80 Ala Glu Tyr Gly Asn Ser Ser Val Phe LeuGlu Asn Ser Thr Phe Asp 85 90 95 Glu Phe Gly His Ser Ile Asn Asp Tyr SerIle Ser Pro Asp Gly Gln 100 105 110 Phe Ile Leu Leu Glu Tyr Asn Tyr ValLys Gln Trp Arg His Ser Tyr 115 120 125 Thr Ala Ser Tyr Asp Ile Tyr AspLeu Asn Lys Arg Gln Leu Ile Thr 130 135 140 Glu Glu Arg Ile Pro Asn AsnThr Gln Trp Val Thr Trp Ser Pro Val 145 150 155 160 Gly His Lys Leu AlaTyr Val Trp Asn Asn Asp Ile Tyr Val Lys Ile 165 170 175 Glu Pro Asn LeuPro Ser Tyr Arg Ile Thr Trp Thr Gly Lys Glu Asp 180 185 190 Ile Ile TyrAsn Gly Ile Thr Asp Trp Val Tyr Glu Glu Glu Val Phe 195 200 205 Ser AlaTyr Ser Ala Leu Trp Trp Ser Pro Asn Gly Thr Phe Leu Ala 210 215 220 TyrAla Gln Phe Asn Asp Thr Glu Val Pro Leu Ile Glu Tyr Ser Phe 225 230 235240 Tyr Ser Asp Glu Ser Leu Gln Tyr Pro Lys Thr Val Arg Val Pro Tyr 245250 255 Pro Lys Ala Gly Ala Val Asn Pro Thr Val Lys Phe Phe Val Val Asn260 265 270 Thr Asp Ser Leu Ser Ser Val Thr Asn Ala Thr Ser Ile Gln IleThr 275 280 285 Ala Pro Ala Ser Met Leu Ile Gly Asp His Tyr Leu Cys AspVal Thr 290 295 300 Trp Ala Thr Gln Glu Arg Ile Ser Leu Gln Trp Leu ArgArg Ile Gln 305 310 315 320 Asn Tyr Ser Val Met Asp Ile Cys Asp Tyr AspGlu Ser Ser Gly Arg 325 330 335 Trp Asn Cys Leu Val Ala Arg Gln His IleGlu Met Ser Thr Thr Gly 340 345 350 Trp Val Gly Arg Phe Arg Pro Ser GluPro His Phe Thr Leu Asp Gly 355 360 365 Asn Ser Phe Tyr Lys Ile Ile SerAsn Glu Glu Gly Tyr Arg His Ile 370 375 380 Cys Tyr Phe Gln Ile Asp LysLys Asp Cys Thr Phe Ile Thr Lys Gly 385 390 395 400 Thr Trp Glu Val IleGly Ile Glu Ala Leu Thr Ser Asp Tyr Leu Tyr 405 410 415 Tyr Ile Ser AsnGlu Tyr Lys Gly Met Pro Gly Gly Arg Asn Leu Tyr 420 425 430 Lys Ile GlnLeu Ile Asp Tyr Thr Lys Val Thr Cys Leu Ser Cys Glu 435 440 445 Leu AsnPro Glu Arg Cys Gln Tyr Tyr Ser Val Ser Phe Ser Lys Glu 450 455 460 AlaLys Tyr Tyr Gln Leu Arg Cys Ser Gly Pro Gly Leu Pro Leu Tyr 465 470 475480 Thr Leu His Ser Ser Val Asn Asp Lys Gly Leu Arg Val Leu Glu Asp 485490 495 Asn Ser Ala Leu Asp Lys Met Leu Gln Asn Val Gln Met Pro Ser Lys500 505 510 Lys Leu Asp Phe Ile Ile Leu Asn Glu Thr Lys Phe Trp Tyr GlnMet 515 520 525 Ile Leu Pro Pro His Phe Asp Lys Ser Lys Lys Tyr Pro LeuLeu Leu 530 535 540 Asp Val Tyr Ala Gly Pro Cys Ser Gln Lys Ala Asp ThrVal Phe Arg 545 550 555 560 Leu Asn Trp Ala Thr Tyr Leu Ala Ser Thr GluAsn Ile Ile Val Ala 565 570 575 Ser Phe Asp Gly Arg Gly Ser Gly Tyr GlnGly Asp Lys Ile Met His 580 585 590 Ala Ile Asn Arg Arg Leu Gly Thr PheGlu Val Glu Asp Gln Ile Glu 595 600 605 Ala Ala Arg Gln Phe Ser Lys MetGly Phe Val Asp Asn Lys Arg Ile 610 615 620 Ala Ile Trp Gly Trp Ser TyrGly Gly Tyr Val Thr Ser Met Val Leu 625 630 635 640 Gly Ser Gly Ser GlyVal Phe Lys Cys Gly Ile Ala Val Ala Pro Val 645 650 655 Ser Arg Trp GluTyr Tyr Asp Ser Val Tyr Thr Glu Arg Tyr Met Gly 660 665 670 Leu Pro ThrPro Glu Asp Asn Leu Asp His Tyr Arg Asn Ser Thr Val 675 680 685 Met SerArg Ala Glu Asn Phe Lys Gln Val Glu Tyr Leu Leu Ile His 690 695 700 GlyThr Ala Asp Asp Asn Val His Phe Gln Gln Ser Ala Gln Ile Ser 705 710 715720 Lys Ala Leu Val Asp Val Gly Val Asp Phe Gln Ala Met Trp Tyr Thr 725730 735 Asp Glu Asp His Gly Ile Ala Ser Ser Thr Ala His Gln His Ile Tyr740 745 750 Thr His Met Ser His Phe Ile Lys Gln Cys Phe Ser Leu Pro 755760 765 7 2366 DNA Homo sapiens misc_feature (1)..(2366) n is anynucleotide 7 gcgnccgctc ccacttcaga ttgaacctaa cgaggtgaca cactcaggagacacaggtgt 60 ggaaacagac ggcagaatgc ctccaaaggt gacttcagag ctgcttcggcagctgagaca 120 agccatgagg aactctgagt atgtgaccga accgatccag gcctacatcatcccatcggg 180 agatgctcat cagagtgagt atattgctcc atgtgactgt cggcgggcttttgtctctgg 240 attcgatggc tctgcgggca cagccatcat cacagaagag catgcagccatgtggactga 300 cgggcgctac tttctccagg ctgccaagca aatggacagc aactggacacttatgaagat 360 gggtctgaag gacacaccaa ctcaggaaga ctggctggtg agtgtgcttcctgaaggatc 420 cagggttggt gtggacccct tgatcattcc tacagattat tggaagaaaatggccaaagt 480 tctgagaagt gccggccatc acctcattcc tgtcaaggag aacctcgttgacaaaatctg 540 gacagaccgt cctgagcgcc cttgcaagcc tctcctcaca ctgggcctggattacacagg 600 catctcctgg aaggacaagg ttgcagacct tcggttgaaa atggctgagaggaacgtcat 660 gtggtttgtg gtcactgcct tggatgagat tgcgtggcta tttaatctccgaggatcaga 720 tgtggagcac aatccagtat ttttctccta cgcaatcata ggactagagacgatcatgct 780 cttcattgat ggtgaccgca tagacgcccc cagtgtgaag gagcacctgcttcttgactt 840 gggtctggaa gccgaataca ggatccaggt gcatccctac aagtccatcctgagcgagct 900 caaggccctg tgtgctgacc tctccccaag ggagaaggtg tgggtcagtgacaaggccag 960 ctatgctgtg agcgagacca tccccaagga ccaccgctgc tgtatgccttacacccccat 1020 ctgcatcgcc aaagctgtga agaattcagc tgagtcagaa ggcatgaggccggctcacat 1080 taaagatgct gttgctctct gtgaactctt taactggctg gagaaagaggttcccaaagg 1140 tggtgtgaca gagatctcag ctgctgacaa agctgaggag tttcgcaggcaacaggcaga 1200 ctttgtggac ctgagcttcc caacaatttc cagtacggga cccaacggcgccatcattca 1260 ctacgcgcca gtccctgaga cgaataggac cttgtccctg gatgaggtgtaccttattga 1320 ctcgggtgct caatacaagg atggcaccac agatgtgacg cggacaatgcattttgggac 1380 ccctacagcc tacgagaagg aatgcttcac atatgtcctc aagggccacatagctgtgag 1440 tgcagccgtt ttcccgactg gaaccaaagg tcaccttctt gactcctttgcccgttcagc 1500 tttatgggat tcaggcctag attacttgca cgggactgga catggtgttgggtctttttt 1560 gaatgtccat gagggtcctt gcggcatcag ttacaaaaca ttctctgatgagcccttgga 1620 ggcaggcatg attgtcactg atgagcccgg gtactatgaa gatggggcttttggaattcg 1680 cattgagaat gttgtccttg tggttcctgt gaagaccaag tataattttaataaccgggg 1740 aagcctgacc tttgaacctc taacattggt tccaattcag accaaaatgatagatgtgga 1800 ttctcttaca gacaaagagt gcgactggct caacaattac cacctgacctgcagggatgt 1860 gattgggaag gaattgcaga aacagggccg ccaggaagct ctcgagtggctcatcagaga 1920 gacgcaaccc atctccaaac agcattaata aatacctccc cggttttgtttttgtaaaat 1980 gctctggagg aaggaagaaa cgtggcagat ccctgacatc tttcccctttcctttccttc 2040 ttccctacct ccccttttta ctttagactt taagaagaac agaaaatcttcttatcctct 2100 ttgatatttt attgcaaaca ctcagtcttt tatgattttt taattgttgagaacaagcca 2160 agaataaaat tgctgcacca gaaggagggt ccctccaaag ttgaacacttggtgaaagga 2220 agatgccccg acttctttgg ccagtgatgg ggaatcagtg agtgctccatgatggtcatg 2280 ttccaggtgc tagtacatca ttcatgatca ccttaatgct catgagactatatttatgat 2340 cagtgaataa aaatgtcaga actgtg 2366 8 623 PRT Homo sapiens8 Met Pro Pro Lys Val Thr Ser Glu Leu Leu Arg Gln Leu Arg Gln Ala 1 5 1015 Met Arg Asn Ser Glu Tyr Val Thr Glu Pro Ile Gln Ala Tyr Ile Ile 20 2530 Pro Ser Gly Asp Ala His Gln Ser Glu Tyr Ile Ala Pro Cys Asp Cys 35 4045 Arg Arg Ala Phe Val Ser Gly Phe Asp Gly Ser Ala Gly Thr Ala Ile 50 5560 Ile Thr Glu Glu His Ala Ala Met Trp Thr Asp Gly Arg Tyr Phe Leu 65 7075 80 Gln Ala Ala Lys Gln Met Asp Ser Asn Trp Thr Leu Met Lys Met Gly 8590 95 Leu Lys Asp Thr Pro Thr Gln Glu Asp Trp Leu Val Ser Val Leu Pro100 105 110 Glu Gly Ser Arg Val Gly Val Asp Pro Leu Ile Ile Pro Thr AspTyr 115 120 125 Trp Lys Lys Met Ala Lys Val Leu Arg Ser Ala Gly His HisLeu Ile 130 135 140 Pro Val Lys Glu Asn Leu Val Asp Lys Ile Trp Thr AspArg Pro Glu 145 150 155 160 Arg Pro Cys Lys Pro Leu Leu Thr Leu Gly LeuAsp Tyr Thr Gly Ile 165 170 175 Ser Trp Lys Asp Lys Val Ala Asp Leu ArgLeu Lys Met Ala Glu Arg 180 185 190 Asn Val Met Trp Phe Val Val Thr AlaLeu Asp Glu Ile Ala Trp Leu 195 200 205 Phe Asn Leu Arg Gly Ser Asp ValGlu His Asn Pro Val Phe Phe Ser 210 215 220 Tyr Ala Ile Ile Gly Leu GluThr Ile Met Leu Phe Ile Asp Gly Asp 225 230 235 240 Arg Ile Asp Ala ProSer Val Lys Glu His Leu Leu Leu Asp Leu Gly 245 250 255 Leu Glu Ala GluTyr Arg Ile Gln Val His Pro Tyr Lys Ser Ile Leu 260 265 270 Ser Glu LeuLys Ala Leu Cys Ala Asp Leu Ser Pro Arg Glu Lys Val 275 280 285 Trp ValSer Asp Lys Ala Ser Tyr Ala Val Ser Glu Thr Ile Pro Lys 290 295 300 AspHis Arg Cys Cys Met Pro Tyr Thr Pro Ile Cys Ile Ala Lys Ala 305 310 315320 Val Lys Asn Ser Ala Glu Ser Glu Gly Met Arg Pro Ala His Ile Lys 325330 335 Asp Ala Val Ala Leu Cys Glu Leu Phe Asn Trp Leu Glu Lys Glu Val340 345 350 Pro Lys Gly Gly Val Thr Glu Ile Ser Ala Ala Asp Lys Ala GluGlu 355 360 365 Phe Arg Arg Gln Gln Ala Asp Phe Val Asp Leu Ser Phe ProThr Ile 370 375 380 Ser Ser Thr Gly Pro Asn Gly Ala Ile Ile His Tyr AlaPro Val Pro 385 390 395 400 Glu Thr Asn Arg Thr Leu Ser Leu Asp Glu ValTyr Leu Ile Asp Ser 405 410 415 Gly Ala Gln Tyr Lys Asp Gly Thr Thr AspVal Thr Arg Thr Met His 420 425 430 Phe Gly Thr Pro Thr Ala Tyr Glu LysGlu Cys Phe Thr Tyr Val Leu 435 440 445 Lys Gly His Ile Ala Val Ser AlaAla Val Phe Pro Thr Gly Thr Lys 450 455 460 Gly His Leu Leu Asp Ser PheAla Arg Ser Ala Leu Trp Asp Ser Gly 465 470 475 480 Leu Asp Tyr Leu HisGly Thr Gly His Gly Val Gly Ser Phe Leu Asn 485 490 495 Val His Glu GlyPro Cys Gly Ile Ser Tyr Lys Thr Phe Ser Asp Glu 500 505 510 Pro Leu GluAla Gly Met Ile Val Thr Asp Glu Pro Gly Tyr Tyr Glu 515 520 525 Asp GlyAla Phe Gly Ile Arg Ile Glu Asn Val Val Leu Val Val Pro 530 535 540 ValLys Thr Lys Tyr Asn Phe Asn Asn Arg Gly Ser Leu Thr Phe Glu 545 550 555560 Pro Leu Thr Leu Val Pro Ile Gln Thr Lys Met Ile Asp Val Asp Ser 565570 575 Leu Thr Asp Lys Glu Cys Asp Trp Leu Asn Asn Tyr His Leu Thr Cys580 585 590 Arg Asp Val Ile Gly Lys Glu Leu Gln Lys Gln Gly Arg Gln GluAla 595 600 605 Leu Glu Trp Leu Ile Arg Glu Thr Gln Pro Ile Ser Lys GlnHis 610 615 620 9 3428 DNA Homo sapiens 9 caccctatcc tacactactaggaacttgca cagtccgcct cgggcagccc aaagctcctc 60 tgcccaccct ggctcccaaaaccctccaaa acaaaagacc agaaaagcac tctccaccca 120 gcagccaaac gcctccttcttgacgccagc ccccaccctc tgtctgctcg agcccaggaa 180 aggcctgaag gaacaggccggggaaggagc cctccctctc tcccttgtcc ctccatccac 240 ccagcgccgg catctggagaccctatggcc cgggctcact ggggctgctg cccctggctg 300 gtcctcctct gtgcttgtgcctggggccac acaaagccac tggaccttgg agggcaggat 360 gtgagaaatt gttccaccaaccccccttac cttccagtta ctgtggtcaa taccacaatg 420 tcactcacag ccctccgccagcagatgcag acccagaatc tctcagccta catcatccca 480 ggcacagatg ctcacatgaacgagtacatc ggccaacatg acgagaggcg tgcgtggatt 540 acaggcttta cagggtctgcaggaactgca gtggtgacta tgaagaaagc agctgtctgg 600 accgacagtc gctactggactcaggctgag cggcaaatgg actgtaattg ggagctccat 660 aaggaagttg gcaccactcctattgtcacc tggctcctca ccgagattcc cgctggaggg 720 cgtgtgggtt ttgaccccttcctcttgtcc attgacacct gggagagtta tgatctggcc 780 ctccaaggct ctaacagacagctggtgtcc atcacaacca atcttgtgga cctggtatgg 840 ggatcagaga ggccaccggttccaaatcaa cccatttatg ccctgcagga ggcattcaca 900 gggagcactt ggcaggagaaagtatctggc gtccgaagcc agatgcagaa gcatcaaaag 960 gtcccgactg ccgtccttctgtcggcgctt gaggagacgg cctggctctt caaccttcga 1020 gccagtgaca tcccctataaccccttcttc tattcctaca cgctgctcac agactcttct 1080 attaggttgt ttgcaaacaagagtcgcttt agctccgaaa ccttgagcta tctgaactcc 1140 agttgcacag gccccatgtgtgtgcaaatc gaggattaca gccaagttcg tgacagcatc 1200 caggcctact cattgggagatgtgaggatc tggattggga ccagctatac catgtatggg 1260 atctatgaaa tgataccaagggagaaactc gtgacagaca cctactcccc agtgatgatg 1320 accaaggcag tgaagaacagcaaggagcag gccctcctca aggccagcca cgtgcgggac 1380 gctgtggctg tgatccggtacttggtctgg ctggagaaga acgtgcccaa aggcacagtg 1440 gatgagtttt cgggggcagagatcgtggac aagttccgag gagaagaaca gttctcctcc 1500 ggacccagtt ttgaaaccatctctgctagt ggtttgaatg ctgccctggc ccactacagc 1560 ccgaccaagg agctgaaccgcaagctgtcc tcagatgaga tgtacctgct ggactctggg 1620 gggcagtact gggacgggaccacagacatc accagaacag tccactgggg caccccctct 1680 gcctttcaga aggaggcatatacccgtgtg ctgataggaa atattgacct gtccaggctc 1740 atctttcccg ctgctacatcagggcgaatg gtggaggcct ttgcccgcag agccttgtgg 1800 gatgctggtc tcaattatggtcatgggaca ggccacggca ttggcaactt cctgtgtgtg 1860 catgagtggc cagtgggattccagtccaac aacatcgcta tggccaaggg catgttcact 1920 tccattgaac ctggttactataaggatgga gaatttggga tccgtctcga agatgtggct 1980 ctcgtggtag aagcaaagaccaagtaccca ggggagctac ctgaccttgt ggtatcattt 2040 gtgccctatg accggaacctcatcgatgtc agcctgctgt ctcccgagca tctccagtac 2100 ctgaatcgct actaccagaccatccgggag aaggtgggtc cagagctgca gaggcgccag 2160 ctactagagg agttcgagtggcttcaacag cacacagagc ccctggccgc cagggcccca 2220 gacaccgcct cctgggcctctgtgttagtg gtctccaccc ttgccatcct tggctggagt 2280 gtctagaggc tccagactctcctgttaacc ctccatctag atggggggct cccttgctta 2340 gctcccctca ccctgcactgaacatacccc aagagcccct gctggcccat tgcctagaaa 2400 cctttgcatt catcctccttctccaagacc tatggagaag gtcccaggcc ccaggaaaca 2460 cagggcttct tggccccagatggcacctcc ctgcaccccg gggttgtata ccacaccctg 2520 ggcccctaat cccaggccccgaaataggaa agccagctag tctcttctct tctgtgatct 2580 cagtaggcct aacctataacctaacacaga ctgctacagc tgctcccctc ccgccaaaca 2640 aagccccaag aaaacaatgcccctaccacc caagggtgcc atggtcccgg gaaaacccaa 2700 cctgtcaccg cgtgttgggcgtaaccagaa ctgttccccc ccaccagggc ttaaaaatcg 2760 cccccacttt ttaaccatcgtccattaacc acctggtggg catagccaga gctgttcgaa 2820 cccagccagg gatgaaaaatcaacccccga catggaaccc atgattccta aacccggggt 2880 aggttccatg ccaagtaacagcagagggag ttaagccata ggaatttggc tgtggagtaa 2940 gagggaatgc ggtgaggcagtgtggaatat gaccctacca gaggttggag aacaaacttg 3000 ggcagccgga acccgtcactattttagatt cctggcattc gaggagccct ttgaactttc 3060 caaagtgcag ccacagctacaatgctgtta aatcctccca catttcttgg atgccccttc 3120 accttgtgtg gacagtgtctggtttcccca ttttacagac aggaaaactg agcttcagac 3180 agggggtggg ctttgcctaaggacacacaa atttggttgg gagttgatgg ggccagatga 3240 gccagcattc cagctgtttcacccttcagc aacatgcaga gtccctgagc ccacctccca 3300 gccctctcct cattctctgaacccactgtg gtgagaagaa tttgctccgg ccaaattggc 3360 cgttagccac ctgggtccacatcctgctaa gacgtttaaa acagcctaac aaagacactt 3420 gcctgtgg 3428 10 493PRT Homo sapiens 10 Val Ser Ile Thr Thr Asn Leu Val Asp Leu Val Trp GlySer Glu Arg 1 5 10 15 Pro Pro Val Pro Asn Gln Pro Ile Tyr Ala Leu GlnGlu Ala Phe Thr 20 25 30 Gly Ser Thr Trp Gln Glu Lys Val Ser Gly Val ArgSer Gln Met Gln 35 40 45 Lys His Gln Lys Val Pro Thr Ala Val Leu Leu SerAla Leu Glu Glu 50 55 60 Thr Ala Trp Leu Phe Asn Leu Arg Ala Ser Asp IlePro Tyr Asn Pro 65 70 75 80 Phe Phe Tyr Ser Tyr Thr Leu Leu Thr Asp SerSer Ile Arg Leu Phe 85 90 95 Ala Asn Lys Ser Arg Phe Ser Ser Glu Thr LeuSer Tyr Leu Asn Ser 100 105 110 Ser Cys Thr Gly Pro Met Cys Val Gln IleGlu Asp Tyr Ser Gln Val 115 120 125 Arg Asp Ser Ile Gln Ala Tyr Ser LeuGly Asp Val Arg Ile Trp Ile 130 135 140 Gly Thr Ser Tyr Thr Met Tyr GlyIle Tyr Glu Met Ile Pro Arg Glu 145 150 155 160 Lys Leu Val Thr Asp ThrTyr Ser Pro Val Met Met Thr Lys Ala Val 165 170 175 Lys Asn Ser Lys GluGln Ala Leu Leu Lys Ala Ser His Val Arg Asp 180 185 190 Ala Val Ala ValIle Arg Tyr Leu Val Trp Leu Glu Lys Asn Val Pro 195 200 205 Lys Gly ThrVal Asp Glu Phe Ser Gly Ala Glu Ile Val Asp Lys Phe 210 215 220 Arg GlyGlu Glu Gln Phe Ser Ser Gly Pro Ser Phe Glu Thr Ile Ser 225 230 235 240Ala Ser Gly Leu Asn Ala Ala Leu Ala His Tyr Ser Pro Thr Lys Glu 245 250255 Leu Asn Arg Lys Leu Ser Ser Asp Glu Met Tyr Leu Leu Asp Ser Gly 260265 270 Gly Gln Tyr Trp Asp Gly Thr Thr Asp Ile Thr Arg Thr Val His Trp275 280 285 Gly Thr Pro Ser Ala Phe Gln Lys Glu Ala Tyr Thr Arg Val LeuIle 290 295 300 Gly Asn Ile Asp Leu Ser Arg Leu Ile Phe Pro Ala Ala ThrSer Gly 305 310 315 320 Arg Met Val Glu Ala Phe Ala Arg Arg Ala Leu TrpAsp Ala Gly Leu 325 330 335 Asn Tyr Gly His Gly Thr Gly His Gly Ile GlyAsn Phe Leu Cys Val 340 345 350 His Glu Trp Pro Val Gly Phe Gln Ser AsnAsn Ile Ala Met Ala Lys 355 360 365 Gly Met Phe Thr Ser Ile Glu Pro GlyTyr Tyr Lys Asp Gly Glu Phe 370 375 380 Gly Ile Arg Leu Glu Asp Val AlaLeu Val Val Glu Ala Lys Thr Lys 385 390 395 400 Tyr Pro Gly Glu Leu ProAsp Leu Val Val Ser Phe Val Pro Tyr Asp 405 410 415 Arg Asn Leu Ile AspVal Ser Leu Leu Ser Pro Glu His Leu Gln Tyr 420 425 430 Leu Asn Arg TyrTyr Gln Thr Ile Arg Glu Lys Val Gly Pro Glu Leu 435 440 445 Gln Arg ArgGln Leu Leu Glu Glu Phe Glu Trp Leu Gln Gln His Thr 450 455 460 Glu ProLeu Ala Ala Arg Ala Pro Asp Thr Ala Ser Trp Ala Ser Val 465 470 475 480Leu Val Val Ser Thr Leu Ala Ile Leu Gly Trp Ser Val 485 490

What is claimed is:
 1. A method of identifying a subject that is susceptible to developing an angioedemic condition during a course of treatment comprising administering one of an ACE inhibitor and a vasopeptidase inhibitor, comprising: (a) providing a biological sample from a subject; (b) determining a dipeptidyl peptidase IV activity in the biological sample; and (c) comparing a dipeptidyl peptidase IV activity in the biological sample to a standard dipeptidyl peptidase IV activity, wherein a 10% or more reduction in the sample activity compared to the standard indicates that the subject is susceptible to developing an angioedema during a course of treatment comprising administering one of an ACE inhibitor and a vasopeptidase inhibitor.
 2. The method of claim 1, wherein the vasopeptidase inhibitor comprises an angiotensin-converting enzyme inhibitor.
 3. The method of claim 1, wherein the vasopeptidase inhibitor comprises a neutral endopeptidase inhibitor.
 4. The method of claim 1, wherein the subject is a human.
 5. The method of claim 1, wherein a 20% or more reduction in the dipeptidyl peptidase IV activity in the biological sample, as compared to the standard dipeptidyl peptidase IV activity indicates that the subject is susceptible to developing an angioedema during a course of treatment comprising administering one of an ACE inhibitor and a vasopeptidase inhibitor.
 6. The method of claim 1, wherein a 30% or more reduction in the dipeptidyl peptidase IV activity in the biological sample, as compared to the standard dipeptidyl peptidase IV activity indicates that the subject is susceptible to developing an angioedema during a course of treatment comprising administering one of an ACE inhibitor and a vasopeptidase inhibitor.
 7. A method of identifying a subject that is susceptible to developing an angioedemic condition during a course of treatment comprising administering one of an ACE inhibitor and a vasopeptidase inhibitor, comprising: (a) providing a biological sample from a subject; (b) determining an aminopeptidase P activity in the biological sample; and (c) comparing an aminopeptidase P activity in the biological sample to a standard aminopeptidase P activity, wherein a 10% or more reduction in the sample activity compared to the standard indicates that the subject is susceptible to developing an angioedema during a course of treatment comprising administering one of an ACE inhibitor and a vasopeptidase inhibitor.
 8. The method of claim 7, wherein the vasopeptidase inhibitor comprises an angiotensin-converting enzyme inhibitor.
 9. The method of claim 7, wherein the vasopeptidase inhibitor comprises a neutral endopeptidase inhibitor.
 10. The method of claim 7, wherein the subject is a human.
 11. The method of claim 7, wherein a 20% or more reduction in the aminopeptidase P activity in the biological sample, as compared to the standard aminopeptidase P activity indicates that the subject is susceptible to developing an angioedema during a course of treatment comprising administering one of an ACE inhibitor and a vasopeptidase inhibitor.
 12. The method of claim 7, wherein a 30% or more reduction in the aminopeptidase P activity in the biological sample, as compared to the standard aminopeptidase P activity indicates that the subject is susceptible to developing an angioedema during a course of treatment comprising administering one of an ACE inhibitor and a vasopeptidase inhibitor.
 13. A method of determining contraindication for administration of one of an ACE inhibitor and a vasopeptidase inhibitor to an individual, comprising: (a) providing a biological sample from a subject; (b) determining a dipeptidyl peptidase IV activity in the biological sample; and (c) comparing a dipeptidyl peptidase IV activity in the biological sample to a standard dipeptidyl peptidase IV activity, wherein administration of the vasopeptidase inhibitor is contraindicated when the dipeptidyl peptidase IV activity in the biological sample is outside the standard dipeptidyl peptidase IV activity range.
 14. The method of claim 13, wherein the vasopeptidase inhibitor is an angiotensin-converting enzyme inhibitor.
 15. The method of claim 13, wherein the vasopeptidase inhibitor is a neutral endopeptidase inhibitor.
 16. A method of determining contraindication for administration of one of an ACE inhibitor and a vasopeptidase inhibitor, comprising: (a) providing a biological sample obtained from a subject; (b) determining an aminopeptidase P activity in the biological sample; and (c) comparing an aminopeptidase P activity in the biological sample to a standard aminopeptidase P activity, wherein administration of the vasopeptidase inhibitor is contraindicated when the aminopeptidase P activity in the biological sample is outside the standard aminopeptidase P activity range.
 17. The method of claim 16, wherein the vasopeptidase inhibitor comprises an angiotensin-converting enzyme inhibitor.
 18. The method of claim 16, wherein the vasopeptidase inhibitor comprises a neutral endopeptidase inhibitor.
 19. A method of screening an individual for compatibility with an administration of one of an ACE inhibitor and a vasopeptidase inhibitor, comprising: (a) providing a biological sample obtained from a subject; (b) determining a dipeptidyl peptidase IV activity in the biological sample; and (c) comparing a dipeptidyl peptidase IV activity in the biological sample to a standard dipeptidyl peptidase IV activity range, wherein administration of the vasopeptidase inhibitor is contraindicated when the sample activity is outside the standard dipeptidyl peptidase IV activity range, and wherein administration of the vasopeptidase inhibitor is indicated when the sample activity is either within or above the standard dipeptidyl peptidase IV activity range.
 20. The method of claim 19, wherein the vasopeptidase inhibitor comprises an angiotensin-converting enzyme inhibitor.
 21. The method of claim 19, wherein the vasopeptidase inhibitor comprises a neutral endopeptidase inhibitor.
 22. A method of screening an individual for compatibility with an administration of one of an ACE inhibitor and a vasopeptidase inhibitor, comprising: (a) providing a biological sample obtained from a subject; (b) determining an aminopeptidase P activity in the biological sample; and (c) comparing an aminopeptidase P activity in the biological sample to a standard aminopeptidase P activity range, wherein administration of a vasopeptidase inhibitor is contraindicated when the sample activity is below the standard aminopeptidase P activity range, and wherein administration of the vasopeptidase inhibitor is indicated when the sample activity is either equal to or above the standard aminopeptidase P activity range.
 23. The method of claim 22, wherein the vasopeptidase inhibitor comprises an angiotensin-converting enzyme inhibitor.
 24. The method of claim 22, wherein the vasopeptidase inhibitor comprises a neutral endopeptidase inhibitor.
 25. A kit for identifying a subject at risk for angioedema during a course of treatment comprising administering one of an ACE inhibitor and a vasopeptidase inhibitor, comprising: (a) a substrate of a dipeptidyl peptidase IV enzyme; (b) a buffer; (c) a reaction stop solution; and (c) a set of instructions comprising information on a standard dipeptidyl peptidase IV activity range.
 26. The kit of claim 25, further comprising a calibration solution for calibration of the reaction.
 27. The kit of claim 25, wherein the substrate comprises Gly-Pro-p-nitroanilide.
 28. A kit for identifying a subject at risk for angioedema during a course of treatment comprising administering one of an ACE inhibitor and a vasopeptidase inhibitor, comprising: (a) an aminopeptidase P enzyme substrate; (b) a dilution buffer; (c) a reaction stop solution; (d) a revelation buffer; and (d) a set of instructions comprising information on a standard aminopeptidase P activity range.
 29. The kit of claim 28, further comprising a calibration solution for calibration of the reaction.
 30. The kit of claim 28, wherein the substrate comprises the peptide Arg-Pro-Pro.
 31. A kit for identifying a subject at risk for angioedema during a course of treatment comprising administering one of an ACE inhibitor and a vasopeptidase inhibitor, comprising: (a) a vasopeptidase inhibitor; and (b) a packaging material comprising information that the vasopeptidase inhibitor is contraindicated for individuals with a serum dipeptidyl peptidase IV enzyme activity outside a standard dipeptidyl peptidase IV activity range.
 32. A kit for identifying a subject at risk for angioedema during a course of treatment comprising administering one of an ACE inhibitor and a vasopeptidase inhibitor, comprising: (a) a vasopeptidase inhibitor; and (b) a packaging material comprising information that the vasopeptidase inhibitor is contraindicated for individuals with a serum aminopeptidease P enzyme activity outside a standard aminopeptidase P activity range.
 33. A kit for identifying a subject at risk for angioedema during a course of treatment comprising administering one of an ACE inhibitor and a vasopeptidase inhibitor, comprising: (a) a vasopeptidase inhibitor; and (b) a packaging material, wherein the packaging material comprises information that the vasopeptidase inhibitor is indicated for individuals with a serum dipeptidyl peptidase IV enzyme activity within a standard dipeptidyl peptidase IV activity range.
 34. A kit for identifying a subject at risk for angioedema during a course of treatment comprising administering one of an ACE inhibitor and a vasopeptidase inhibitor, comprising: (a) a vasopeptidase inhibitor; and (b) a packaging material, wherein the packaging material comprises information that the vasopeptidase inhibitor is indicated for individuals with a serum aminopeptidase P enzyme activity within a standard aminopeptidase P activity range.
 35. A method of marketing a vasopeptidase inhibitor comprising providing information about a diagnostic test adapted to identify a subject that is susceptible to angioedema as a result of taking the vasopeptidase inhibitor during a course of treatment comprising administering one of an ACE inhibitor and a vasopeptidase inhibitor.
 36. The method of claim 35, wherein the vasopeptidase inhibitor comprises an angiotensin-converting enzyme inhibitor.
 37. The method of claim 35, wherein the vasopeptidase inhibitor comprises a neutral endopeptidase inhibitor.
 38. The method of claim 35, wherein the diagnostic test comprises detecting dipeptidyl peptidase IV activity in a biological sample derived from the subject.
 39. The method of claim 35, wherein the diagnostic test comprises detecting aminopeptidase P activity in a biological sample derived from the subject.
 40. The method of claim 35, wherein the subject is a human. 